Liposomes, emulsions, and methods for cryotherapy

ABSTRACT

A method and system in accordance with a particular embodiments of the technology includes applying a substance onto skin of a human subject. The applied substance can include a freezing point depressant and a liposome, an oil-in-water emulsion, a water-in-oil emulsion, or an oil-in-oil emulsion and can be configured to protect or target tissue. The substance and a surface of the skin can be cooled using the applicator to treat acne and other skin conditions.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 15/498,867, filed Apr. 27, 2017, now pending, which claims thebenefit of and priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application No. 62/334,330, filed May 10, 2016, which areincorporated herein by reference in their entirety.

INCORPORATION BY REFERENCE

The following U.S. Patent Applications and U.S. Patents are incorporatedherein by reference in their entireties:

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U.S. Patent Publication No. 2014/0005760 entitled “CRYOPROTECTANT FORUSE WITH A TREATMENT DEVICE FOR IMPROVED COOLING OF SUBCUTANEOUSLIPID-RICH CELLS”;

U.S. Patent Publication No. 2007/0270925 entitled “ METHOD AND APPARATUSFOR NON-INVASIVELY REMOVING HEAT FROM SUBCUTANEOUS LIPID RICH CELLSINCLUDING A COOLANT HAVING A PHASE TRANSITION TEMPERATURE”;

U.S. Patent Publication No. 2009/0118722 entitled “METHOD AND APPARATUSFOR COOLING SUBCUTANEOUS LIPID-RICH CELLS OR TISSUE”;

U.S. Patent Publication No. 2008/0287839 entitled “METHOD OF ENHANCEDREMOVAL OF HEAT FROM SUBCUTANEOUS LIPID-RICH CELLS AND TREATMENTAPPARATUS HAVING AN ACTUATOR”;

U.S. Patent Publication No. 2013/0079684 entitled “METHOD OF ENHANCEDREMOVAL OF HEAT FROM SUBCUTANEOUS LIPID-RICH CELLS AND TREATMENTAPPARATUS HAVING AN ACTUATOR”;

U.S. Pat. No. 8,285,390 entitled “MONITORING THE COOLING OF SUBCUTANEOUSLIPID-RICH CELLS, SUCH AS THE COOLING OF ADIPOSE TISSUE”;

U.S. Pat. No. 9,408,745 entitled “MONITORING THE COOLING OF SUBCUTANEOUSLIPID-RICH CELLS, SUCH AS THE COOLING OF ADIPOSE TISSUE”;

U.S. Patent Publication No. 2013/0116758 entitled “MONITORING THECOOLING OF SUBCUTANEOUS LIPID-RICH CELLS, SUCH AS THE COOLING OF ADIPOSETISSUE”;

U.S. Pat. No. 8,523,927 entitled “SYSTEM FOR TREATING LIPID-RICHREGIONS”;

U.S. Patent Publication No. 2014/0067025 entitled “SYSTEM FOR TREATINGLIPID-RICH REGIONS”;

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U.S. Patent Publication No. 2009/0018625 entitled “MANAGING SYSTEMTEMPERATURE TO REMOVE HEAT FROM LIPID-RICH REGIONS”;

U.S. Patent Publication No. 2009/0018626 entitled “USER INTERFACES FOR ASYSTEM THAT REMOVES HEAT FROM LIPID-RICH REGIONS”;

U.S. Patent Publication No. 2009/0018627 entitled “SECURE SYSTEM FORREMOVING HEAT FROM LIPID-RICH REGIONS”;

U.S. Pat. No. 8,275,442 entitled “TREATMENT PLANNING SYSTEMS AND METHODSFOR BODY CONTOURING APPLICATIONS”;

U.S. Patent Publication No. 2013/0158440 entitled “TREATMENT PLANNINGSYSTEMS AND METHODS FOR BODY CONTOURING APPLICATIONS”;

U.S. patent application Ser. No. 12/275,002 entitled “APPARATUS WITHHYDROPHILIC RESERVOIRS FOR COOLING SUBCUTANEOUS LIPID-RICH CELLS”;

U.S. patent application Ser. No. 12/275,014 entitled “APPARATUS WITHHYDROPHOBIC FILTERS FOR REMOVING HEAT FROM SUBCUTANEOUS LIPID-RICHCELLS”;

U.S. Pat. No. 8,676,338 entitled “COMBINED MODALITY TREATMENT SYSTEMS,METHODS AND APPARATUS FOR BODY CONTOURING APPLICATIONS”;

U.S. Patent Publication No. 2014/0316393 entitled “COMBINED MODALITYTREATMENT SYSTEMS, METHODS AND APPARATUS FOR BODY CONTOURINGAPPLICATIONS”;

U.S. Pat. No. 8,603,073 entitled “SYSTEMS AND METHODS WITHINTERRUPT/RESUME CAPABILITIES FOR COOLING SUBCUTANEOUS LIPID-RICHCELLS”;

U.S. Patent Publication No. 2013/0245731 entitled “SYSTEMS AND METHODSWITH INTERRUPT/RESUME CAPABILITIES FOR COOLING SUBCUTANEOUS LIPID-RICHCELLS”;

U.S. Pat. No. 8,702,774 entitled “DEVICE, SYSTEM AND METHOD FOR REMOVINGHEAT FROM SUBCUTANEOUS LIPID-RICH CELLS”;

U.S. Patent Publication No. 2014/0257443 entitled “DEVICE, SYSTEM ANDMETHOD FOR REMOVING HEAT FROM SUBCUTANEOUS LIPID-RICH CELLS”;

U.S. Patent Publication No. 2014/0257443 entitled “ COMPOSITIONS FOR USEWITH A SYSTEM FOR IMPROVED COOLING OF SUBCUTANEOUS LIPID-RICH TISSUE”;

U.S. Publication No. 2012/0239123 entitled “DEVICES, APPLICATION SYSTEMSAND METHODS WITH LOCALIZED HEAT FLUX ZONES FOR REMOVING HEAT FROMSUBCUTANEOUS LIPID-RICH CELLS ”;

U.S. Pat. No. 6,041,787 entitled “USE OF CRYOPROTECTIVE AGENT COMPOUNDSDURING CRYOSURGERY”;

U.S. Pat. No. 6,032,675 entitled “FREEZING METHOD FOR CONTROLLED REMOVALOF FATTY TISSUE BY LIPOSUCTION”;

U.S. Pat. No. 9,314,368 entitled “HOME-USE APPLICATORS FORNON-INVASIVELY REMOVING HEAT FROM SUBCUTANEOUS LIPID-RICH CELLS VIAPHASE CHANGE COOLANTS, AND ASSOCIATED DEVICES, SYSTEMS AND METHODS”;

U.S. Publication No. 2011/0238051 entitled “HOME-USE APPLICATORS FORNON-INVASIVELY REMOVING HEAT FROM SUBCUTANEOUS LIPID-RICH CELLS VIAPHASE CHANGE COOLANTS, AND ASSOCIATED DEVICES, SYSTEMS AND METHODS”;

U.S. Pat. No. 9,545,523 entitled “MULTI-MODALITY TREATMENT SYSTEMS,METHODS AND APPARATUS FOR ALTERING SUBCUTANEOUS LIPID-RICH TISSUE”;

U.S. Patent Publication No. 2014/0277302 entitled “TREATMENT SYSTEMSWITH FLUID MIXING SYSTEMS AND FLUID-COOLED APPLICATORS AND METHODS OFUSING THE SAME”;

U.S. Patent Publication No. 2015/0216720 entitled “TREATMENT SYSTEMS,METHODS, AND APPARATUSES FOR IMPROVING THE APPEARANCE OF SKIN ANDPROVIDING FOR OTHER TREATMENTS”;

U.S. Patent Publication No. 2015/0216816 entitled “COMPOSITIONS,TREATMENT SYSTEMS AND METHODS FOR IMPROVED COOLING OF LIPID-RICHTISSUE”;

U.S. Patent Publication No. 2015/0216719 entitled “TREATMENT SYSTEMS ANDMETHODS FOR TREATING CELLULITE AND FOR PROVIDING OTHER TREATMENTS”;

U.S. patent application Ser. No. 14/662,181 entitled “TREATMENT SYSTEMS,DEVICES, AND METHODS FOR COOLING TARGETED TISSUE”;

U.S. patent application Ser. No. 14/710,407 entitled “TREATMENT SYSTEMSWITH ADJUSTABLE GAP APPLICATORS AND METHODS FOR COOLING TISSUE”;

U.S. Patent Publication No. 2016/0054101 entitled “TREATMENT SYSTEMS,SMALL VOLUME APPLICATORS, AND METHODS FOR TREATING SUBMENTAL TISSUE”;

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U.S. Patent Publication No. 2016/0089550 entitled “TREATMENT SYSTEMS,METHODS, AND APPARATUSES FOR ALTERING THE APPEARANCE OF SKIN”;

U.S. Patent Publication No. 2017/0007309 entitled “TREATMENT SYSTEMS ANDMETHODS FOR AFFECTING GLANDS AND OTHER TARGETED STRUCTURES”;

U.S. Patent Publication No. 2016/0317346 entitled “SYSTEMS AND METHODSFOR MONITORING COOLING OF SKIN AND TISSUE TO IDENTIFY FREEZE EVENTS”;

U.S. patent application Ser. No. 15/271,121 entitled “TRANSCUTANEOUSTREATMENT SYSTEMS, COOLING DEVICES, AND METHODS FOR COOLING NERVES”;

U.S. patent application Ser. No. 15/296,853 entitled “VASCULAR TREATMENTSYSTEMS, COOLING DEVICES, AND METHODS FOR COOLING VASCULAR STRUCTURES”;

U.S. patent application Ser. No. 15/400,885 entitled“TEMPERATURE-DEPENDENT ADHESION BETWEEN APPLICATOR AND SKIN DURINGCOOLING OF TISSUE”;

U.S. Provisional Patent Application Ser. No. 62/334,213 entitled “SKINFREEZING SYSTEMS FOR TREATING ACNE AND SKIN CONDITIONS”;

U.S. Provisional Patent Application Ser. No. 62/334,317 entitled“HYDROGEL SUBSTANCES AND METHODS OF CRYOTHERAPY”;

U.S. Provisional Patent Application Ser. No. 62/334,337 entitled“PERMEATION ENHANCERS AND METHODS OF CRYOTHERAPY”; and

U.S. Provisional Patent Application Ser. No. 62/297,054 entitled“COOLING CUP APPLICATORS WITH CONTOURED HEADS AND LINER ASSEMBLIES”.

TECHNICAL FIELD

The present disclosure relates generally to systems for cooling tissue.In particular, several embodiments are directed to treatment systems,methods, and substances for controllably cooling tissue to treat acne orother conditions.

BACKGROUND

Exocrine glands found in the skin have a role in maintaining skinhealth, including lubricating, waterproofing, cleansing and/or coolingthe skin or hair follicles of the body by excreting water-based, oilyand/or waxy substances through skin pores or hair follicles.Overproduction and/or oversecretion of these substances by certainexocrine glands, such as sebaceous glands and sudoriparous glands (e.g.,sweat glands), can cause unappealing skin disorders that have proveddifficult to treat. For example, overproduction of sebum, a waxysubstance produced and secreted by sebaceous glands, can lead to theformation of comedones (e.g., blackheads, whiteheads, etc.) and otherinflammatory conditions of the skin associated with acne (e.g., inflamedpapules, pustules, nodules, etc.), which can potentially lead toscarring of the skin. Overproducing sebaceous glands associated withhair follicles are mostly found in highly visible regions of the body,such as along the face, neck, upper chest, shoulders and back.

Hyperhidrosis is a condition associated with excessive sweating causedby the overproduction and secretion of sweat from sweat glands in theskin of mammals. Excessive sweating from eccrine sweat glands, which aredistributed almost all over the body, can cause discomfort andembarrassment. For example, focal hyperhidrosis can occur on the palmsof the hands, soles of the feet, face and scalp. Apocrine sweat glands,particularly in the axilla (i.e., armpits), have oil-producing cellsthat can contribute to undesirable odor.

Treatments for these and other skin and tissue conditions are oftenineffective, non-lasting, and/or have undesirable side-effects.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Many aspects of the present invention can be better understood withreference to the following drawings. Identical reference numbersidentify similar elements or acts. The sizes and relative positions ofelements in the drawings are not necessarily drawn to scale.

FIG. 1 is a schematic cross-sectional view of the skin, dermis, andsubcutaneous tissue of a subject.

FIG. 2 is a schematic cross-sectional view of the skin, dermis, andsubcutaneous tissue of the subject in FIG. 1 after treating sebaceousglands.

FIG. 3 is a partially schematic, isometric view of a treatment systemfor non-invasively treating targeted structures in a human subject'sbody in accordance with an embodiment of the technology.

FIG. 4 is a cross-sectional view of a conduit of the treatment system ofFIG. 3 .

FIG. 5 is a flow diagram illustrating a method for treating a subject'sskin in accordance with an embodiment of the technology.

FIG. 6 is a plot of temperature versus time for a treatment involvingminimal or no skin supercooling when an applicator is initially placedon a patient's skin to initiate a freeze event.

FIG. 7 is a plot of temperature versus time for a treatment involvingsubstantial skin cooling.

FIG. 8 is a flow diagram illustrating a method for treating a subject'sskin in accordance with an embodiment of the technology.

FIGS. 9A-9C show stages of a method for preparing a treatment site inaccordance with an embodiment of the disclosed technology.

FIG. 10A shows patterned hydrogel suitable for cryotherapy in accordancewith an embodiment of the disclosed technology.

FIGS. 10B and 10C are plots of propylene glycol concentration versuslength for patterned hydrogels.

FIGS. 11A and 11B are side views of hydrogel substances with icenucleating regions in accordance with embodiments of the technology.

FIG. 12A shows an emulsifier or surfactant with a hydrophilic head and ahydrophobic tail.

FIG. 12B shows an agent entrapped by emulsifiers.

FIGS. 13A and 13B show an oil-in-water emulsion and a water-in-oilemulsion.

FIG. 14 is a table with melting/freezing point temperatures for fats.

FIGS. 15A and 15B show test results performed on skin in accordance withsome embodiments of the disclosed technology.

FIG. 16 shows a treatment cycle temperature profile and a temperatureresponse measured by a temperature sensor at an applicatorsurface-tissue interface.

FIG. 17 shows the temperature of the applicator surface lowered at aconstant rate and then held at a generally constant value in accordancewith an embodiment of the technology.

FIG. 18 is a plot of temperature versus time showing an exemplarytreatment cycle for controlled supercooling and for controlled freezingof tissue by lowering the skin temperature in accordance with anembodiment of the technology.

FIGS. 19A-19E are cross-sectional views of an applicator applied to atreatment site and thermal modeling.

FIGS. 20A-20F illustrate stages of one method of freezing tissue withoutsupercooling.

FIG. 21 is a plot of temperature versus time for freezing skin multipletimes in accordance with an embodiment of the disclosed technology.

FIG. 22A shows an unfrozen liquid coupling media that can serve as aninsulator for ice inoculation of skin.

FIG. 22B shows the coupling media of FIG. 22A with a shifted temperatureprofile.

FIG. 23 is a plot of temperature versus propylene glycol (PG)concentration in water in accordance with an embodiment of the disclosedtechnology.

FIGS. 24A-24F show stages of a method for supercooling the skin and theninitiating a freeze event in accordance with an embodiment of thedisclosed technology.

FIG. 25 is a plot of temperature versus time for supercooling andfreezing tissue.

FIG. 26 is a plot of temperature versus time for a procedure that cyclestwo times to supercool tissue and then triggers a freeze.

FIGS. 27A-27C show an applicator and a coupling media at various stagesduring a procedure.

FIG. 28 shows an applicator applied to a treatment site in accordancewith an embodiment of the disclosed technology.

FIG. 29 is a plot of temperature versus time for a temperature profilefor triggering ice nucleation by activating an ice nucleator.

FIG. 30 shows an applicator and an ice nucleating agent (INA) applied toa treatment site in accordance with an embodiment of the disclosedtechnology.

FIG. 31 shows a plot of temperature versus time for delivering an INAfor nucleation.

FIGS. 32A-32D are IR images showing stages of a process using hydrogelto freeze supercooled tissue.

FIGS. 33A-33D are IR images showing tissue freeze inoculation usingcombined materials.

FIGS. 34A and 34B are cross-sectional views of an applicator applied toa treatment site in accordance with some embodiments of the disclosedtechnology.

FIG. 35 is a plot of temperature versus time for triggering a freezeagent using a hydrogel.

FIG. 36 shows an applicator positioned to produce a controlled freeze ina coupling material in accordance with an embodiment of the disclosedtechnology.

FIG. 37 shows an applicator with an external nucleating elementconfigured to initiate a freeze event at a location external to anapplicator-hydrogel interface.

FIG. 38 is a cross-sectional view of an applicator applied to atreatment site and capable of providing energy-based activation.

FIG. 39 is a plot of temperature versus time for supercooling skin priorto initiating a freeze event.

FIG. 40 is a plot of temperature versus time where, after supercoolingand before freezing, a temperature of the applicator is adjusted to warmthe epidermis.

FIG. 41 shows a plot of temperature versus time for a cooling protocoland three cross-sectional views of an applicator and skin tissue andtemperature distributions.

FIG. 42 shows a temperature profile in the epidermis/dermis andtemperature distribution for one treatment protocol.

FIG. 43 shows a temperature profile into the epidermis/dermis for onetreatment protocol.

FIG. 44 shows a stage in one method of creating an intradermal tissuefreeze using an injectable substance.

FIG. 45 is a flow diagram illustrating a method for preparing andfreezing tissue in accordance with an aspect of the present technology.

FIG. 46 is a schematic block diagram illustrating subcomponents of acomputing device in accordance with an embodiment of the disclosure.

DETAILED DESCRIPTION A. Overview

The present disclosure describes treatment systems and substances forimproving the appearance, function and health of tissue and forperforming other treatments. Several of the details set forth below areprovided to describe the following examples and methods in a mannersufficient to enable a person skilled in the relevant art to practice,make and use them. Several of the details and advantages describedbelow, however, may not be necessary to practice certain examples andmethods of the technology. Additionally, the technology may includeother examples and methods that are within the scope of the technologybut are not described in detail.

Various aspects of the technology are directed to cooling a surface of apatient's skin to produce a cooling event (e.g., a partial freeze event,a total freeze event, etc.) that affects tissue, cells, structures,appendages, or targeted features. Systems disclosed herein can targetglands (e.g., exocrine glands, sebaceous glands, sweat glands,sudoriparous glands, etc.), structures in the skin (e.g., hairfollicles, superficial nerves, etc.), and/or layer(s) of tissue (e.g.,dermal layer, epidermal layer, subcutaneous layer, sub-layer(s) of theepidermis, dermis, subcutaneous, etc.). In some embodiments, the coolingevent reduces or limits overproduction and/or oversecretion of exocrineglands to treat comedones and/or other inflammatory conditions of theskin associated with acne, such as inflamed papules, pustules, nodules,etc. For example, the cooling event can cause an effective amount ofthermal injury to glands to reduce or limit overproduction and/oroversecretion by those glands to reduce or eliminate acne or other skinconditions. The cooling event can include freezing a region of thedermal layer containing the targeted exocrine glands without affectingnon-targeted tissue. Treatment applicators can be configured for usealong the face, neck, upper chest, shoulders, back, and other treatmentsites and can target specific layers in the skin, subcutaneous tissue,specific structures, particular cells, etc.

In some embodiments, a method for treating a subject's skin includesapplying a coupling media to the skin. The coupling media can include afreezing point depressant and either a liposome, an oil-in-wateremulsion, a water-in-oil emulsion, or an oil-in-oil emulsion containingthe freezing point depressant to enhance delivery of the freezing pointdepressant to the skin. The coupling media and a surface of the skin canbe cooled with an applicator to a temperature below 0° C. to treat theskin.

In one embodiment, a method for treating a subject's skin includesapplying a coupling media to a surface of the subject's skin such thatan ice nucleating agent contained in the applied coupling media does notdirectly contact epidermal cells for a period of time. An applicator isapplied to the subject's skin. The coupling media and the surface of theskin are cooled to lower a temperature of the surface of the skin below0° C. to cause a freeze event initiated by the ice nucleating agent soas to freeze the skin. In one procedure, an applied is held against theskin while the applicator cools the skin.

In another embodiment, a system for treating a subject includes anapplicator and a controller. The applicator can be configured to reducea temperature of a target region beneath a surface of the subject's skinto reduce the temperature of target tissue from a natural bodytemperature to a temperature for freezing the target tissue. Thecoupling media can include a freezing point depressant and either aliposome, an oil-in-water emulsion, a water-in-oil emulsion, oroil-in-oil emulsion containing the freezing point depressant to enhancedelivery of the freezing point depressant to the skin. The controllercan be programmed to cause the applicator to cool the coupling media anda surface of the skin to a temperature below 0° C. to freeze the skinfor a period of time.

At least some of the embodiments disclosed herein can be forcosmetically beneficial treatments. As such, some treatment proceduresmay be for the sole purpose of altering a treatment site to conform to acosmetically desirable look, feel, size, shape or other desirablecosmetic characteristic or feature. Accordingly, cosmetic procedures canbe performed without providing any or minimal therapeutic effect. Forexample, some treatment procedures may be directed to goals, such as thereduction of acne, that do not include restoration of health, physicalintegrity, or the physical well-being of a subject. In some embodiments,methods can target skin irregularities, wrinkles, and sebaceous glandsto treat acne; sweat glands to treat hyperhidrosis; hair follicles toinjure and remove hair; or other targeted cells to change a subject'sappearance or address a condition. Treatments may have therapeuticoutcomes (whether intended or not), such as, psychological benefits,alteration of body hormone levels (by the reduction of adipose tissue),etc. Various aspects of the methods disclosed herein can includecosmetic treatment methods for achieving a cosmetically beneficialalteration of a portion of tissue within the target region. Suchcosmetic methods can be administered by a non-medically trained person.The methods disclosed herein can also be used to (a) improve theappearance of skin by tightening the skin, improving skin tone andtexture, eliminating or reducing wrinkles, increasing skin smoothness,thickening the skin, (b) improve the appearance of cellulite, and/or (c)treat sebaceous glands, hair follicles, and/or sweat glands.

At least some embodiments of the technology include producing one ormore controlled freeze events. The location and extent of freezing canbe controlled to produce a therapeutic or cosmetic effect. Nucleationinitiators, nucleation inhibitors, and/or treatment substances can beused before, during, and/or after the freeze event. The nucleationinitiators can include, without limitation, ice nucleation agents,injectable substances (e.g., saline, ice slurries, etc.), energy thatpromotes ice nucleation, or other initiators that affect freezing.Nucleation inhibitors can include, without limitation, cryoprotectantsolutions, freeze temperature depressants, and/or heaters.

According to one aspect of the technology, a subject's skin is loweredto below its melting/freezing point (“melting point”). The skintemperature is monitored to control an amount of non-freezing effects.An ice crystal contacts skin to cause a freeze event in the skin. Theskin can be monitored to control an amount of freeze treatment. The skincan also be monitored to detect any further non-freeze effects, freezeeffects, or thaw effects to precisely and predictably control an overalllevel of treatment. Skin preparation techniques can be utilized toenhance absorption of the substance into the skin by abrading and/orscrapping of the epidermis. Example substances include thermal couplinggels, cryoprotectant solutions, and/or ice nucleating agents that may beincorporated into or part of a hydrogel material, a liposome, anemulsion, a nano-emulsion, nanoparticle mixture or solution, and/orcombinations thereof. Nano-emulsions and nanoparticles may be desirablesince their small size makes them suitable to being absorbed into theepidermis and dermis by traveling along hair follicle apertures and skinpore apertures. A cryoprotectant can be used to enhance the amount ofnon-freezing treatment to be delivered prior to any freeze event becausethe cryoprotectant can allow significant supercooling of the skin priorto initiating a freeze event. In one embodiment, an ice nucleating agentis utilized to reliably and predictably form ice crystals.

An applicator can predictably freeze targeted tissue or structures byproducing a freeze event that occurs in an expected way. For example,tissue can be cooled to start a freeze event at an anticipated time(e.g., at a particular time or within an expected period of time),propagate the freeze at a desired rate, achieve a desired extent offreezing, or the like. Treatment parameters can be selected based on thedesired predictability of the freeze event. For example, the skinsurface can be cooled to produce a freeze event at least 80%, 85%, 90%,or 95% of the time in a typical patient. This provides a predictablefreeze. If a freeze event does not occur, the skin can be warmed andcooled again to produce a freeze event.

One advantage of freezing is that for a given amount of desired tissuedamage, a procedure that produces freezing can take considerably lesstime than a procedure which does not involve freezing. This is becausewith freezing, cell walls are damaged.

Damage to tissue due to freezing and cooling is mainly dependent on, forexample, cooling rate, end temperature, holding time (unfrozen and/orfrozen), and thawing rate. These variables can be controlled to achievethe desired cryoinjury to target tissue.

Tissue damage at the cellular scale is known to occur due tointracellular (IIF) and extracellular ice (EIF) formation. Cryoinjurydue to IIF can be accomplished by inducing irreversible damage to thetissues and by necrosis destroying cell organelles and membranes fromthe inside. Cryoinjury due to extracellular ice formation is mainly dueto hyperosmolarity in extracellular space and dehydration of the cellsbecause of the extracellular ice. These processes provoke direct celldeath or programmed cell death (e.g., apoptosis of the cells).

In order to accomplish tissue injury, a sufficient end low temperaturecan be reached. Individual tissues and cells may have differentsusceptibility to cold. Consequently, lethal temperatures can vary amongdifferent components of the skin. Multiple cycles of a treatmenttemperature protocol should increase efficacy as well.

Holding time in a frozen state enhances cryogenic tissue injurymechanisms. As ice crystals grow in size during a holding time period,the more they will enhance injury due to IIF and/or EIF.

Thawing is a destructive factor facilitating recrystallization (icecrystal restructuring), namely, crystals growing bigger, and rehydrationof cells causing membrane disruption and cell death.

For skin, cold can affect the blood microcirculation which can inducereversible or irreversible vascular changes. During cooling there isvasoconstriction of blood vessels which in some temperature treatmentprotocols may provoke the occurrence of stasis and tissue ischaemia.During freezing, there may be damage to the endothelium of blood vesselsand other cellular injury due to EIF and IIF. Vasoconstrictionfacilitates hypoxia, a state in which cells release vasodilatationcytokines which after thawing enhance refractory vasodilatation andreperfusion injury. Reperfusion also facilitates inflammatory andperivascular oedema of tissues.

Additionally, partially or totally frozen tissue has a higher thermalconductivity and a lower specific heat than unfrozen tissue. The thermalconductivity continues to increase and the specific heat continues todecrease as additional tissue is frozen. This change in thermalproperties can result in enhanced efficiency (e.g., a factor of four toeight improvement in cooling efficiency) as compared to a treatmentwhich does not involve freezing, even when the treatment temperatures ofthe non-freezing treatment with supercooling are similar to the freezingtreatment temperature. Accordingly, with freezing, the depth ofpenetration of cooling into the skin and surrounding tissue can besignificantly faster than without freezing.

Some embodiments are directed to treating tissue below the skin orsublayers or sub-thicknesses of the skin, such as the epidermis, dermis,subdermis, subcutaneous, and sub-layers thereof to treat wrinkles, finelines, pores, moles, freckles, port wine stains, and other vascularissues, acne, or the like. Additionally or alternatively, treatments canbe performed to rejuvenate skin, resurface skin, address skin colorationissues, block pain, etc., and to affect targets, such as appendages,cellular elements, or combinations thereof. Appendages that can betreated include, without limitation, hair follicles, sebaceous glands,sweat glands, arrector pili, nerves, blood vessels, etc. Cellularelements that can be treated include, without limitation, corneocytes,keratinocytes, melanocytes, sebocytes, fibroblasts, blood cells,collagen, elastin fibers, etc. The systems and methods disclosed hereinare useful for treating the targets and conditions disclosed herein.

References throughout this specification to “one example,” “an example,”“one embodiment,” or “an embodiment” mean that a particular feature,structure, or characteristic described in connection with the example isincluded in at least one example of the present technology. Thus, theoccurrences of the phrases “in one example,” “in an example,” “oneembodiment,” or “an embodiment” in various places throughout thisspecification are not necessarily all referring to the same example. Theheadings provided herein are for convenience only and are not intendedto limit or interpret the scope or meaning of the technology.

B. Treatment Sites

FIG. 1 is a schematic cross-sectional view of the skin, dermis, andsubcutaneous tissue of a subject. A subject's skin 10 includes thedermis 12 located between the epidermis 14 and the subcutaneous layer16. The dermis 12 includes sebaceous glands 17 that produce sebum, awaxy substance secreted for moisturizing the skin and hair. Acne is askin condition typically characterized by excess sebum that may plughair follicles and/or pores. The level of sebum production may varybetween individuals and may vary by body location depending on thenumber and sizes of the sebaceous glands. Sebum can flow along thehealthy hair follicle 20 to moisturize the hair 23 and/or epidermis 14.When the sebaceous glands 17 produce excess sebum, it can collect and/orbecome trapped in hair follicles. Overproduction and/or entrapment ofsebum can lead to formation of comedones (e.g., blackheads, whiteheads,etc.), as well as other inflammatory conditions of the skin associatedwith acne (e.g., inflamed papules, pustules, nodules, etc.). In someindividuals, inflamed follicles and pores can become infected and thecondition can potentially lead to scarring of the skin. The illustratedhair follicle 22 is clogged with excess sebum to form a pimple or redspot. Other medical conditions associated with overactive sebaceousglands 17 include sebaceous cysts, hyperplasia and sebaceous adenoma.Non-medical, but cosmetically unappealing, conditions associated withoveractive sebaceous glands include oily skin and/or oily hair (e.g., onthe scalp).

Another skin condition is hyperhidrosis. Hyperhidrosis is characterizedby abnormal sweating due to high secretion levels of sweat glands 26.Eccrine sweat glands are controlled by the sympathetic nervous systemand regulate body temperature. When an individual's body temperaturerises, eccrine sweat glands secrete sweat (i.e., water and othersolutes) that flows through a gland tubule 28. The sweat can evaporatefrom the skin surface to cool the body. Apocrine sweat glands (notshown) secrete an oil-containing sweat into hair follicles 20. Theaxilla (e.g., armpit) and genital regions often have a highconcentration of apocrine sweat glands. Hyperhidrosis occurs when sweatglands produce and secrete sweat at levels above that required forregulation of body temperature, and the condition can be generalized orlocalized (i.e., focal hyperhidrosis) to specific body parts (e.g.,palms of hands, soles of feet, brow, scalp, face, underarms, etc.).

FIG. 2 is a schematic cross-sectional view of the skin and a side viewof a treatment device in the form of a thermoelectric applicator 104(“applicator 104”) applied to the skin to treat acne, hyperhidrosis, andother skin conditions by freezing the skin. The applicator 104 cancontrollably produce predictable freeze events to avoid under treatment,overtreatment, and/or undesirable side effects, such as damage tonon-targeted tissue or structures. Freezing skin to damage tissue can bedifficult to control, thus often results in undertreatment orovertreatment. This is because freezing of skin and tissue below theskin tend to be somewhat random and unpredictable. Water in biologicaltissue, such as the skin 10, has the tendency to remain in a liquidstate for a certain period of time, even if its temperature is loweredbelow its melting/freezing point, a phenomenon termed “supercooling.”The terms “supercooling,” “supercooled,” and “supercool” refer to acondition in which a material is at a temperature below itsfreezing/melting point but is still in an unfrozen or mostly unfrozenstate. It can be somewhat unpredictable whether a freeze event will everoccur, and if so when the freeze event will occur during the treatmentand how long tissue will be in a frozen state. In addition, it is oftenvery difficult to control freeze-thaw parameters, such as a freezingrate, target freeze temperatures, duration of freeze events, and awarming rate. These freeze-thaw parameters need to be controlled toachieve predictable therapeutic outcomes. It may be difficult to controlfreeze-thaw parameters, thus making it difficult to control an amount oftreatment. When the amount of treatment is too large, undesirable sideeffects can occur, such as undesired skin pigmentation changes, and whenit is too small, insufficient efficacy can result. This lack of controlalso can make it difficult to target certain tissue for treatment and tominimize treatment of other specific non-targeted tissue.

The applicator 104 can accurately target tissue while minimizing orlimiting effects on non-targeted tissue. It has been discovered thatwhen an ice crystal contacts skin 10 of the subject at a temperaturewhich is below its phase transition temperature (e.g., melting/freezingtemperature) and in a supercooled state, a freeze event can beimmediately triggered in the skin. The ice crystal can thus be used topredictably control initiation of the freeze event. Once the freezeevent is triggered, it can rapidly propagate through the volume ofsupercooled tissue. The heat of fusion released during freezing may takethe bulk tissue out of its supercooled state, and thereafter thepartially frozen skin may prevent non-frozen tissue from reentering asupercooled state. Additionally, the heat of fusion in some procedures,the period of time from the beginning of a freeze event in supercooledtissue to the point where the tissue is largely no longer in asupercooled state may be 1 second, 2 seconds, 3 seconds, 5 seconds, 10seconds, or another suitable period of time. The time period forsupercooling can depend on the target location, volume of targetedtissue, supercooled tissue volume, temperature profile, tissuecharacteristics (e.g., water content of tissue), and/or additives (e.g.,compositions, energy, etc.) that may be used as part of the procedure.Because freeze propagation rates may be strongly dependent on thesupercool temperature, the temperature of the supercooled tissue can bedecreased or increased to increase or decrease, respectively, freezepropagation rates.

The applicator 104 can be used to precisely control a start time of thefreeze event, an amount of damage caused by an initial freeze event(e.g., by controlling an amount of supercooling created prior toinitiating the freeze event), a duration of the freeze event (e.g., bycontrolling a temperature of an applicator), and thawing rate (e.g.,start of thaw cycle, etc.). The timing of freeze events can be preciselycontrolled by controlling the generation of the ice crystal and when theice crystal comes in contact with supercooled skin so that freeze eventscan be produced “on command,” and this control allows for specializedtreatment methods to be implemented to controllably and effectivelytreat a range of tissue while controlling and/or limiting damage totissue. In addition, additives can be used to manage freeze events atvarying optimum temperatures to target tissue at varying skin depthswhile controlling tissue damage, extent of injury to non-targetedtissue, etc. By controlling when and how to freeze, treatment procedurescan target certain tissue without targeting other tissue while alsocontrolling a level of treatment of targeted tissue and effects tonon-targeted tissue.

FIG. 2 shows the skin 10 after a freeze-induced injury has affected thesebaceous glands 17 to reduce or limit sebum production. Skin 10 hasbeen frozen to controllably disrupt or injure the sebaceous glands 17 orassociated structures which can be an effective treatment for acne.Although the effect to the sebaceous glands 17 is shown while theapplicator 104 is applied to the skin 10, it may take a relatively longperiod of time (e.g., days, weeks, months, etc.) for the glands to bereduced after treatment. The sebum production level of the two sebaceousglands 17 in FIG. 2 , along the hair follicle 22, has been substantiallyreduced to inhibit clogging to minimize, reduce, or eliminate acne. Thesweat gland 26 can also be targeted. For example, the applicator 104 canproduce a partial or total freeze event, non-freezing cooling event, orsupercooling event to affect the sweat gland 26 and/or gland tubule 28in a region of the skin located along the hands, armpits, or otherlocations with excessive sweat. Other structures in the dermis or otherlayers of tissue can be targeted. Accordingly, the cold, associated witha controlled freeze generated by the applicator 104, can generallyreduce/relieve inflammation associated with acne and be an importanttreatment pathway. Any and all these pathways of treatment areencompassed by at least one of the embodiments of the technologydisclosed herein.

In some embodiments, a temperature-controlled surface 111 of theapplicator 104 can be cooled to affect target structures, such asglands, hair follicles, nerves (e.g., superficial nerves), or one ormore layers of tissue (e.g., dermal layer, epidermal layer, subcutaneouslayer, sub-layer(s) of the epidermis, dermis, and/or subcutaneous layer,etc.). To treat acne, the surface of the subject's skin can be cooled toproduce a temperature at or below −15° C., −10° C., −5° C., 0° C., 5°C., 10° C., 15° C., or 20° C. and to produce either a cooling non-freezeevent or a freeze event in a targeted portion of the skin. Localizedfreeze events can be generated to affect targeted structures whileminimizing, limiting, or substantially preventing thermal injuries tonon-targeted tissue, structures, etc. Substances used with theapplicator 104 can include cryoprotectants, nucleating agents,liposomes, emulsions, hydrogels, combinations thereof, or the like.Mechanical energy (e.g., massaging), ultrasound energy, radiofrequency(RF) energy, and/or freeze initiators can control a freeze event by, forexample, initiating, promoting, and/or inhibiting freezing. In someprocedures, ultrasound energy is delivered to supercooled tissue totrigger freezing in the tissue. Radiofrequency energy can be used towarm tissue to isolate freezing to a target region. Freeze initiatorscan be used to initiate a freeze event in the tissue or freeze event inanother substance that ultimately causes freezing in the tissue. Examplefreeze initiators include, but are not limited to, one or more water icecrystals, cryoprobes, or substances that rapidly freeze to producefreeze events. Freeze events can include partially or completelyfreezing liquids or lipids proximate to or within cells, and/orstructures, to destroy, reduce, disrupt, modify, or affect targetedfeatures. The characteristics of the cooling event or freeze event canbe controlled to manage thermal injury. Such characteristics include,without limitation, the amount of cooling or freezing, density anddistribution of ice crystals, freezing rate, or the like.

Cryotherapy can affect, without limitation, glandular function,structures of glands (e.g., gland portions, duct portions, etc.), numberof glands, sizes of glands, and/or number and/or sizes of cells. Thefreeze event can be maintained for a period of time long enough toelicit a desired result. In some embodiments, for treating exocrineglands, a subject's skin can be cooled to produce a partial freeze eventthat destroys, reduces, disrupts, modifies, or affects cells orstructures of exocrine glands or the supporting anatomical features(e.g., ducts, pores, hair follicles, etc.). The level of freezing can becontrolled to limit unwanted tissue damage, such as damage tonon-targeted tissue, excess damage to targeted tissue (e.g., to avoidexcess damage to targeted tissue), and so forth. The skin surface can becontinuously or periodically cooled or heated to increase or decrease,respectively, the number and/or sizes of ice crystals at the targetregion. In one procedure, the tissue can be kept in a supercooled statefor longer than, for example, about 1 second, 5 seconds, 10 seconds, 20seconds, 30 seconds, 1 minute, several minutes, or other time periodselected to allow the tissue to reach a steady state temperature anddesired width, length, and depth of a tissue volume which is in asupercooled state. Once tissue is frozen, it can be kept in a partiallyor totally frozen state for longer than about, for example, about 1second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 1 minute, severalminutes, or other time period selected to achieve desired effects, whilereducing or limiting undesired effects, such as frostbite or necrosis.

The applicator 104 can include one or more elements 167 for detectingcooling events, freezing events, supercooling, and so forth. The thermaldevice 109 can be controlled based on the output from the element 167 tocool a temperature-controlled surface 111, which, in turn, cools thepatient's skin. The element 167 can include one or more temperaturesensors, pressure sensors, detectors, combinations thereof, or the like.Alternatively, separate sensors can be used to monitor the treatmentsite.

C. Treatment Systems

FIG. 3 is a partially schematic, isometric view of a treatment systemfor non-invasively treating targeted structures in a body of a humansubject 101 in accordance with an embodiment of the technology. Thetreatment system 100 can include the applicator 104, a connector 103,and a base unit 106. The applicator 104 can be applied to acne-proneregions to reduce the temperature of lipid-producing cells residing inor at least proximate to sebaceous glands (e.g., glandular epithelialcells) to lower the amount of secreted sebum and thereby eliminate,reduce, or limit acne. The applicator 104 can also cool sweat glands andassociated structures to treat hyperhidrosis and can perform othertreatment procedures. The size and configuration of the applicator 104can be selected based on the treatment site.

The connector 103 can be a cord that provides energy, fluid, and/orsuction from the base unit 106 to the applicator 104. The base unit 106can include a fluid chamber or reservoir 105 (illustrated in phantomline) and a controller 114 carried by a housing 125 with wheels 126. Thebase unit 106 can include a refrigeration unit, a cooling tower, athermoelectric chiller, heaters, or any other devices capable ofcontrolling the temperature of coolant in the fluid chamber 105 and canbe connectable to an external power source and/or include an internalpower supply 110 (shown in phantom line). The power supply 110 canprovide electrical energy (e.g., a direct current voltage) for poweringelectrical elements of the applicator 104. A municipal water supply(e.g., tap water) can be used in place of or in conjunction with thefluid chamber 105. In some embodiments, the system 100 can include apressurization device 117 that can provide suction and can include oneor more pumps, valves, and/or regulators. Air pressure can be controlledby a regulator located between the pressurization device 117 and theapplicator 104. If the vacuum level is too low, tissue may not beadequately (or at all) held against the applicator 104, and theapplicator 104 may tend to move along the patient's skin. If the vacuumlevel is too high, undesirable patient discomfort and/or tissue damagecould occur. A vacuum level can be selected based on the characteristicsof the tissue and desired level of comfort. In other embodiments, theapplicator 104 does not use a vacuum.

An operator can control operation of the treatment system 100 using aninput/output device 118 of the controller 114. The input/output device118 can display the state of operation of the applicator 104 andtreatment information. In some embodiments, the controller 114 can becommunicatively coupled to and exchange data with the applicator 104 viaa wired connection or a wireless or an optical communication link andcan monitor and adjust treatment based on, without limitation, one ormore treatment profiles and/or patient-specific treatment plans, such asthose described, for example, in commonly assigned U.S. Pat. No.8,275,442, which is incorporated by reference in its entirety. In someembodiments, the controller 114 can be incorporated into the applicator104 or another component of the system 100.

Upon receiving input to start a treatment protocol, the controller 114can cycle through each segment of a prescribed treatment plan. Segmentsmay be designed to supercool tissue, to nucleate supercooled tissue, tofreeze tissue, to thaw tissue, to warm tissue, and so on. In so doing,the power supply 110 and the fluid chamber 105 can provide power andcoolant to one or more functional components of the applicator 104, suchas thermoelectric coolers (e.g., TEC “zones”), to begin a cooling cycleand, in some embodiments, to activate features or modes, such asvibration, massage, vacuum, etc.

The controller 114 can receive temperature readings from temperaturesensors, which can be part of the applicator 104 or proximate to theapplicator 104, the patient's skin, a patient protection device, etc. Itwill be appreciated that while a target region of the body has beencooled or heated to the target temperature, in actuality that region ofthe body may be close, but not equal to, the target temperature, e.g.,because of the body's natural heating and cooling variations. Thus,although the system 100 may attempt to heat or to cool tissue to thetarget temperature or to provide a target heat flux, a sensor maymeasure a sufficiently close temperature or heat flux. If the targettemperature or the flux has not been reached, power can be increased ordecreased to change heat flux to maintain the target temperature or“set-point” selectively to affect targeted tissue. The treatment sitecan be continuously or intermittently evaluated by monitoring variousparameters. The skin can be continuously monitored to detect itstemperature to determine whether it is in a frozen state, an unfrozenstate, or other state.

In some procedures, the applicator 104 can achieve a level or amount ofsupercooling at a suitable temperature below, for example, −15° C., −10°C., −5° C., or 0° C. After achieving a predetermined level ofsupercooling, the applicator 104 can automatically start a freeze event.The freeze event can be detected and/or monitored using the applicator104 or separate device. A level of treatment can be controlled followinginitiation and/or completion of the freeze event. One or more postfreeze protocols can be performed to thaw or otherwise thermally affecttissue to allow treatment to be specifically tailored to effectivelytreat certain targets, and to not treat or minimize treatment ofnon-targeted tissue. For example, post-freeze protocols can be used toinhibit, limit, or substantially minimize permanent thermal injuries. Insome embodiments, post-freeze protocols can include gradually or rapidlywarming non-targeted and targeted tissue.

FIG. 4 is a cross-sectional view of the connector 103 taken along line4-4 of FIG. 3 in accordance with at least some embodiments of thetechnology. The connector 103 can be a multi-line or multi-lumen conduitwith a main body 179 (e.g., a solid or hollow main body), a supply fluidline or lumen 180 a (“supply fluid line 180 a”), and a return fluid lineor lumen 180 b (“return fluid line 180 b”). The main body 179 may beconfigured (via one or more adjustable joints) to “set” in place for thetreatment of the subject. The supply and return fluid lines 180 a, 180 bcan be tubes made of polyethylene, polyvinyl chloride, polyurethane,and/or other materials that can accommodate circulating coolant, such aswater, glycol, synthetic heat transfer fluid, oil, a refrigerant, and/orany other suitable heat conducting fluid. In one embodiment, each fluidline 180 a, 180 b can be a flexible hose surrounded by the main body179. Referring now to FIGS. 3 and 4 , coolant can be continuously orintermittently delivered to the applicator 104 via the supply fluid line180 a and can circulate through the applicator 104 to absorb heat. Thecoolant, which has absorbed heat, can flow from the applicator 104 backto the base unit 106 via the return fluid line 180 b. For warmingperiods, the base unit 106 (FIG. 3 ) can heat the coolant such that warmcoolant is circulated through the applicator 104. The connector 103 canalso include one or more electrical lines 112 (FIG. 4 ) for providingpower to the applicator 104 and one or more control lines 116 forproviding communication between the base unit 106 and the applicator104. To provide substances, the connector 103 can include one or moretubes or lines 119 for substances to be delivered by the applicator 104.The substances can include coupling media, INAs, solutions (e.g.,cryoprotectant solutions), or the like.

FIG. 5 is a flow diagram illustrating a method 140 for treating asubject's skin in accordance with an embodiment of the technology.Generally, the subject's skin can be cooled below a freezing temperatureof fluid in the skin. One or more ice crystals can be moved into contactwith the skin to create a predictable freeze event therein. The time ofcontact between the ice crystals and skin can be controlled to achievedesired freezing. Details of the method 140 are discussed below.

At block 142, the skin can be cooled to lower the temperature of theskin below a freezing temperature of fluid in the skin. For example, thetemperature of the skin can be lowered to a first temperature that ismore than 3° C., 5° C., 7° C., 9° C., 10° C., or 11° C. below themelting/freezing temperature of fluid in the skin and can be maintainedfor a first period of time. After the first period of time expires, theskin temperature can be lowered to a second temperature that is lowerthan the first temperature so as to create an ice crystal. In otherembodiments, the first temperature can be maintained at a constanttemperature while creating an ice crystal by, for example, altering thecomposition of a coupling media. The coupling media can freeze and causeice nucleation in the tissue.

At block 144 of FIG. 5 , an ice crystal can contact the subject's skinto inoculate the skin upon contact and to create a predictable freezeevent therein. The ice crystal can be formed externally by anapplicator. Alternatively, a catheter or other device can introduce theice crystal into the subject such that the ice crystal physicallycontacts tissue to be initially frozen. In some procedures, an agent canbe cooled and then diluted to produce one or more ice crystals therein.For example, the agent can include a cryoprotectant for protectingtissue. The concentration of cryoprotectant in the agent can be dilutedto raise a melting/freezing point of the diluted agent to a value abovea temperature of the cryoprotectant so that formation of the ice crystaldoes not require that the skin temperature be lowered to a value belowthe melting/freezing temperature of the cryoprotectant.

At block 146, a time of contact between the ice crystal and the skin canbe controlled. A user can hold the applicator against the skin surfacewhile the ice crystal contacts the skin surface. Upon completion of acontact period, the system can notify the subject or operator to removethe applicator from the subject. The applicator can be pulled off thesubject to stop the crystal contact. Alternatively, the applicator canbe warmed to melt the ice crystal at the completion of the desiredcontact period. The temperature of the applicator can be controlled toset the length of ice crystal/tissue contact, as well as the length ofthe freeze event by detecting the freeze event and further controllingwhen the temperature of the skin is raised to a temperature above theice crystal's melting point to stop the freeze event.

In some treatments, the method 140 can include lowering a temperature ofa subject's skin below a melting/freezing point or temperature of targettissue of the skin. The applicator 104 can monitor cooling of the skinusing the sensor so that freezing therein does not occur. The amount ofnon-freezing cooling treatment delivered to the skin can be controlledso that targeted tissue of the skin reaches a predetermined first levelat block 142. After the targeted tissue reaches the predetermined firstlevel, the skin is frozen (block 144). The sensor can be used toidentify and monitor the freeze event. An ice crystal can come intointimate contact with the supercooled skin during the supercoolingperiod and prior to the time when the beginning of a freeze event isdesired to occur, without adverse effects. After the supercooling periodhas elapsed to create a predetermined first level of supercooling, theice crystal(s) can be brought into contact with the skin to initiate thefreeze event, and damage associated with the initial freeze event can belargely proportional to the level or extent of supercooling. The freezeevent can be maintained for any desired period of time, and after thefreeze event, additional freeze events can further affect the tissue.The amount of freezing/cooling treatment delivered to the skin can becontrolled so that it reaches a predetermined second level. In sometreatments, the ice crystal is used to cause freezing of skin in thefirst level of the supercooled state. The predetermined second level,when combined with the first level, can be selected to provide atherapeutically effective amount of thermal injury.

A shallow skin treatment can include contacting the subject's skin withice crystals while the skin has a bulk temperature just slightly belowits melting/freezing point, such as by 0.2° C., 0.5° C., 1° C., 2° C.,or 3° C. There may be minimal to no significant skin supercooling, sothat the initial freeze event is small (e.g., a fraction of the tissueinitially frozen will be small) and relatively small tissue can befrozen when the initial freeze event occurs. Accordingly, initial tissuedamage can be predominately located in the epidermis and upper layer ofthe dermis, with deeper layers, such as subdermal, fat and muscletissue, being largely unaffected. As such, treatments can be performedon acne-prone regions where damage to subcutaneous tissue may beproblematic and undesired. Once the freeze event occurs, additionaltissue in the skin will not enter a supercooled state because icecrystals in the skin can inhibit or prevent further supercooling.Further additional incremental cooling can result in predictableincremental freezing, and if minimal depth of treatment is desired, atissue thaw protocol can be started immediately or very soon followingthe freeze event.

The system 100 can also perform deeper treatments, including aggressiveand deeper skin treatments, by supercooling targeted tissue and thencontacting the targeted tissue with an ice crystal to trigger a freezeevent. The supercooled tissue can include epidermal tissue, dermaltissue, subcutaneous tissue and can be cooled as much as by 4° C., 5°C., 6° C., 7° C., 8° C., 9° C., 10° C., 12° C., 15° C., 17° C., 20° C.,25° C., 30° C., or 35° C. and for a significant period of time, such as30 seconds or 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25 or more minutes. Thetemperature and treatment period can allow for variable and controlledlevels of skin supercooling prior to initiating a freeze event. Anoverall level of treatment can also include multiple treatments, each ofwhich individually delivers a dose which is less than a total dose oftreatment to ultimately be delivered. For example, after an initial skinsupercooling and freeze event has been performed at a given treatmentsite, device software can be programmed to repeat the supercooling andfreeze event cycle a second time, optionally after a tissuerewarming/thaw step between cycles. Temperatures and the treatmentperiod for the second cycle can be the same as those of the first cycleor different. Additional treatment cycles could also be delivered. Inthis example, it would not be necessary to move the applicator betweencycles, and the cycles could optionally be separated by a tissuerewarming/thaw step. As another alternative, an additional treatment atany given site could be performed later in the patient procedure afteran applicator has treated other tissue sites. Still another alternativeis that an additional treatment could be delivered during a separatepatient procedure performed either later the same day as the firsttreatment, or the next day or several days or a week later, and theprocedure could be repeated on a regular basis if desired (e.g., everyday, every other day, every week, every month, etc.). Any number ofdesired follow-on treatments can be performed to achieve sufficient anddesired overall levels of tissue treatment to create a desired tissueresponse.

The method 140 can be used to perform the treatments disclosed herein,such as the treatments discussed in connection with FIGS. 1 and 2 . Afreeze event can cause disruption of sebaceous glands to affect sebumproduction (e.g., decrease or limit sebum production). The period oftime of contact (e.g., time of contact between the subject's skin andice crystals and/or between the subject's skin and the cooling surfaceof the applicator) can be selected to achieve the desired thermal injuryto the sebaceous glands. The systems, components, and acts disclosedherein can be mixed and matched, as discussed in connection withexamples 1-4 below.

Example 1

Ice crystals can be formed along an applicator, using temperatureprogramming. Water (e.g., droplets of water, a layer containing water,etc.) can be disposed on the applicator surface (e.g., surface 111 inFIG. 2 ), and the temperature of the applicator surface can be loweredto about −20° C., −15° C., −12° C., or another suitable temperature forgenerating one or more ice crystals based on water freezing at or belowits melting/freezing temperature of 0° C. In some procedures, theapplicator can be prepped to have ice crystals on its exterior surfaceto cause a skin freeze event to occur once the applicator contacts theskin surface. For example, one or more ice crystals carried by theapplicator can physically contact the skin to initiate a freeze event inthe skin. In other procedures, the ice crystals can physically contactand trigger a freeze event in a coupling media on the skin surface. Whenthe coupling media freezes, it can cause freezing of the skin surfaceand subsequent freeze propagation through deeper tissue. In otherprocedures, the coupling media can be absorbed into the skin, and theabsorbed coupling media can freeze to cause freezing of the skin.

Tissue can be slowly or rapidly rewarmed as soon as practicable after afreeze event has occurred to limit, reduce, or prevent damage andadverse side effects associated with the freeze event. After freezingbegins, the skin can be slowly or rapidly warmed as soon as possible tominimize or limit damage to the epidermis. In other procedures, the skinis partially or completely frozen for a predetermined period of time andthen warmed. According to one embodiment, the applicator 104 of FIG. 2can warm shallow tissue using, for example, thermoelectric elements inthe device 109. Thermoelectric elements can include Peltier devicescapable of operating to establish a desired temperature (or temperatureprofile) along the surface 111. In other embodiments, the applicator 104has electrodes that output radiofrequency energy for warming tissue.

Absorption enhancers, cryoprotectant agents, INAs, and coupling mediacan be delivered via liposomes, hydrogels, emulsions, or the like.Absorption enhancers can increase permeation to affect uptake of, forexample, water, INAs, cryoprotectants, etc. Skin can be warmed before orduring exposure to applied substances to increase uptake into theepidermis, with minimal or limited increased uptake into the dermis dueto the dermal-epidermal junction barrier. The characteristics of thetissue can be affected by mechanically altering the subject's skin.These characteristics can include absorption characteristics, thermalcharacteristics, or the like. For a treatment which does not includefreezing and only cooling or supercooling, it is desirable to increasean uptake of a cryoprotectant into the skin to provide maximumprotection against the possibility of a non-intended freeze occurring.For a treatment which is to include freezing, it is desirable toincrease an uptake of an INA and/or water to increase the possibility ofa freeze event being initiated and being initiated at a desired time,and to increase a level of cryoinjury.

FIG. 6 is a plot of applicator temperature versus time for a treatmentinvolving minimal or no skin supercooling when an applicator isinitially placed on a subject to initiate a freeze event in accordancewith an embodiment of the disclosed technology. A freeze event can beinitiated upon placement of a frozen applicator surface on the skin. Forexample, the applicator surface (e.g., surface 111 shown in FIG. 2 ) canbe cooled to a temperature of −15° C. to form ice crystals thereon.After the temperature of the applicator surface is raised at a desiredrate to a suitable temperature for placement on the subject, theapplicator surface can be applied to the treatment site. For example,the applicator surface can be warmed at a rate of 0.4° C./s, 0.5° C./s,or 0.6° C./s to a temperature of about −4° C., −3° C., −2° C., −1° C.,0° C., etc. The skin surface, targeted tissue, etc. can be maintained ata temperature of about −3° C., −2° C., −1° C., 0° C., or 1° C.

The applicator can be kept in thermal contact with the skin surface fora first treatment period (e.g., 2 minutes, 2.5 minutes, 3 minutes, etc.,with 2.5 minutes being shown in FIG. 6 ) to cool the skin from aninitial temperature (e.g., 33° C.) to a lower temperature (e.g., −4° C.,−3° C., −2° C., −1° C., 0° C.). The applicator surface can then belowered at a desired rate to a temperature for inducing a freeze event.The freeze event (indicated by an “*” in FIG. 6 ) can occur while theapplicator surface is cooled at a rate of about 0.2° C./s, 0.25° C./s,0.3° C./s, or other desired rate. The applicator surface can be held ata temperature of about −8° C. for a second treatment period (e.g., 20seconds, 30 seconds, 40 seconds, etc.). The skin surface temperature canbe slightly higher than the temperature of the applicator surface, sothe temperature of the applicator surface can be selected to keep thetarget tissue frozen for a desired freeze period.

After completion of the freeze period, the applicator and skintemperature can be rapidly raised to a normal temperature, such as roomtemperature or above. In some procedures, the applicator can be warmedat a rate of about 1° C./s, 2° C./s, 2.5° C./s, 3° C./s, or other rateselected to thaw frozen tissue. FIG. 6 shows the temperature of theapplicator raised at a rate of about 2.5° C./s. The thawed tissue caninclude epidermal tissue, dermal tissue, subcutaneous tissue, and/orother tissue. After the tissue is warmed for a warm period, anothercryotherapy procedure can be performed at the same or difference siteusing the same or different treatment parameters.

Example 2

A substance can be applied to either the skin, the applicator, or both,and can be used to generate ice crystals. The substance can be acoupling media with one or more cryoprotectant agents and can be appliedwhen it is initially at a temperature above its melting point, which canbe several degrees below 0° C. and lower than a melting/freezing pointof fluid in the skin tissue. The melting/freezing point of the appliedsubstance can be in a therapeutic skin supercool treatment temperaturerange or other suitable temperature range. After a predetermined amountof skin supercooling has occurred, the temperature of the appliedsubstance can be lowered to a value below its melting point ortemperature to create ice crystals therein to initiate the freeze eventin the skin.

Cryoprotectant agents can comprise propylene glycol, glycerol,polyethylene glycol, combinations thereof, or other biocompatibleagents. In some embodiments, the substance is a cryoprotectant solutionwith a cryoprotectant agent mixed with water to provide a desiredmelting/freezing point. The concentration of the cryoprotectant agentcan be increased to lower the melting/freezing point of the substance.By controlling a concentration of the cryoprotectant, characteristics ofthe substance (e.g., melting point, spontaneous freezing point, etc.)can be controlled, thus enabling ice crystal generation at/below anydesired temperatures while inhibiting or preventing ice crystalgeneration at/above certain temperatures. INAs can be incorporated intothe substance to, for example, provide predictable initiation of freezeevents once the temperature of the substance is lowered below themelting/freezing point of the INA.

FIG. 7 is a plot of applicator temperature versus time for a treatmentinvolving substantial skin cooling in accordance with an embodiment ofthe disclosed technology. The applicator and skin can be cooled at adesired rate (e.g., 0.5° C./s, 1° C./s, 2° C./s, etc.) to a supercooledtemperature (e.g., −8° C., −10° C., −12° C.). A freeze event in the skincan be initiated after a supercooling period of about 3 minutes, 4minutes, or 5 minutes at the supercooled temperature, illustrated as−10° C. During this period, the skin surface and cooled applicatorsurface can be at substantially the same temperature. A cryoprotectantcoupling media can help limit thermal injuries to non-targeted tissueand can be disposed at the applicator-skin interface. In someembodiments, the cryoprotectant coupling media is about 25% by weight orvolume propylene glycol (PG) and about 75% by weight or volume water andhas a melting or freezing temperature of about −11° C. The compositionof the coupling media can be adjusted to increase or decrease itsmelting/freezing point. After the supercooling period, a temperature ofthe applicator is further lowered to initiate a freeze event. FIG. 7shows initiation of the freeze event in the skin while the applicatorand skin surface are cooled from about −10° C. to about −18° C. Thelevel of freeze in the tissue can be maintained while the applicatorsurface and skin surface are held at a temperature of about −18° C. for10 seconds prior to rapid rewarming.

Ice crystals can be generated by diluting a precooled coupling media toraise its melting/freezing point. The applied substance can be a 25% byvolume PG cryoprotectant solution with a melting temperature of −11° C.Tissue can be supercooled to a desired temperature (e.g., −8° C., −10°C., −12° C., etc.). After the desired amount of supercooling hasoccurred, the freeze event can be initiated by further lowering thetemperature of the applied substance (e.g., “diving” the temperature) toa temperature of about −18° C. so as to freeze the substance. Instead offurther lowering the temperature, or “diving,” a freeze event can beinitiated at a target freeze temperature (e.g., −10° C.) or at a highertemperature by injecting cold water or another substance into theapplied substance at a predetermined location to locally dilute thecryoprotectant concentration to a level whereat the melting point of thecoupling media is higher than the target freeze temperature. Themelting/freezing point of the coupling media can be, for example, closeto −1° C., −0.5° C., or 0° C. so that ice crystals form in the dilutedsubstance and initiate a freeze event in the skin. Dilution can be with100% water, water doped with an INA, or another substance, therebyproviding consistent and predictable freezes at temperatures as warm as,for example, −1° C., −2° C., or −3° C. Alternatively, a water and icemixture or a water, ice, and INA mixture can be injected to providefreezes at about a desired temperature (e.g., −1° C., −0.5° C., etc.).This method can be used in conjunction with substantial skinsupercooling to initiate a freeze event at relatively warm temperatures,for example −1° C., −2° C., −3° C., −4° C., or −5° C., by pre-warmingthe skin after the supercooling period at a lower temperature (e.g., −8°C., −10° C., or −12° C.) has elapsed, which can significantly reduce orprevent harm to non-targeted tissue, such as the epidermis, as comparedto a treatment where the freeze event is started at a lower temperature,such as −10° C. or even lower (e.g., −18° C. when “diving” is utilizedto initiate the freeze event).

Example 3

Energy can be used to manage ice crystal formation. When aqueouscoupling medias are lowered below their melting/freezing points and arein a supercooled state, ultrasound can induce ice crystal formation inthe skin and/or a freeze event in the coupling media whether or not thecoupling media is only slightly or significantly supercooled. Althoughdelivering ultrasound can obviate INAs, ultrasound and INAs can be usedtogether. Ultrasound has been used to form ice crystal in aqueouscoupling agents. For example, a dental cleaning ultrasound probeoperated at about 20 kHz and about 25 W forms ice crystals in couplingagents. In another example, a non-dental ultrasound probe operated atabout 20 kHz and 1 W forms ice crystals. Ultrasound with otherparameters can be selected based on desired ice crystal formation and/orgrowth.

Example 4

After tissue is in a supercooled state, a freeze event triggering orpromoting substance can be injected into or near the target region. Thesubstance can be partially frozen ice or a water slurry solution thatgenerates an immediate freeze event. In some embodiments, the epidermiscan be rewarmed to a temperature close to 0° C. prior to the freezeevent, and an injection of saline ice water slurry into the dermis caninitiate the controlled freeze under the epidermis. Needles, catheters,or injection devices can be introduced into the subject to inject thesubstance. FIG. 2 shows an optional catheter 149 that can be introducedinto the subject. Once an end portion of the catheter 149 is positionedin the skin 10, the catheter 149 can deliver an ice crystal, ice slurry,or suitable substance in the tissue. The catheter 149 can be used toinitiate freeze events at any number of treatment sites.

Various combinations of steps in examples 1-4 can be combined. Toenhance or maximize freeze injury in the dermis while limiting orminimizing side effects associated with freezing in the epidermis,contact between an ice crystal and the tissue can be delayed until adesired level of skin supercooling is achieved. A volume of target skincan be substantially supercooled and then contacted by ice crystals tomaximize freeze injury to the skin while minimizing side effects. Alarge amount of prior supercooling can maximize an amount of tissuedamage that occurs during the initial freeze event, and can allownon-targeted tissue to be rewarmed to inhibit, limit, or substantiallyprevent thermal injury to that non-targeted tissue. The epidermis can benon-targeted tissue that can be immediately or quickly re-warmed afterthe freeze event in the targeted tissue, such as the dermis. Warming canlimit or minimize an amount of time the epidermis is in a frozen state.This is in contrast to a treatment method whereby little or nosupercooling is employed. In this latter case, to obtain a therapeuticlevel of treatment equivalent to the former case (which utilizessubstantial supercooling and substantial fractional freezing during theinitial freeze event, since cooling is delivered “top down,” via thesurface of the skin), the epidermal tissue needs to be maintained in afrozen state longer after the freeze event is initiated, which canexacerbate damage to non-targeted epidermal tissue.

To restrict freezing injury to mostly upper skin layers whilesignificantly sparing deeper tissue from significant injury, icecrystals can contact the skin immediately or very soon after the skintemperature is lowered below the skin's melting/freezing point. Limitedsuperficial epidermal freezes can be achieved with minimal injury todermal, fat and muscle layers, especially when the duration of thefreeze event is kept relatively short. In some facial procedures, thefreeze injury can be limited to the skin to avoid any appreciablereduction of subcutaneous tissue or underlying muscle which form asupport structure for the skin.

FIG. 8 is a flow diagram illustrating a method 150 for treating asubject's skin in accordance with an embodiment of the technology.Generally, coupling media can be applied to the treatment site. Thetreatment site can be cooled, and a freeze event can be initiated to atleast partially freeze tissue. An early stage of the method 150 caninclude coupling a heat-exchanging surface of an applicator to thesubject's skin. The heat-exchanging surface can be atemperature-controlled surface (see, e.g., surface 111 of FIG. 2 ) of aheat-exchanging plate with internal thermal elements (e.g.,thermoelectric elements, fluid elements, etc.) or external thermalelements (e.g., thermal elements mounted to the backside of theheat-exchanging plate). In some embodiments, the temperature-controlledsurface can be an interface layer, a dielectric layer, or the like.Additionally or alternatively, a vacuum or suction force can be used topositively couple the patient's skin to the temperature-controlledsurface. Coupling the temperature-controlled surface to the subject'sskin can also include providing a substance to the patient's skin as isdescribed herein and in commonly assigned U.S. Patent Publication No.2007/0255362. Details of the method 150 are discussed below.

At block 152, the treatment site can be prepared by, for example,mechanically, chemically, or otherwise altering the skin. Mechanicalalteration can be achieved by brushing or scraping the skin surfaceintermittently or continuously for a period of time, such as about 30seconds, 1 minute, 2 minutes, 3 minutes, or a suitable length of timeselected based on the desired amount of surface cleaning, permeation,and/or exfoliation (e.g., exfoliation of the stratum corneum). In otherembodiments, permeability of the skin can be adjusted by clearing poresin the stratum corneum, producing and/growing vacuoles (e.g., vacuolesin the epidermis below the stratum corneum), combinations thereof, orthe like. In some treatments, an adhesive strip can be applied to andremoved from the skin to remove uppermost layers of the epidermis, cleanthe treatment site, increase permeability of the skin, or otherwiseprepare the treatment site. The uppermost layers of the epidermis aredryer than lower layers, so when the uppermost layers are removed, theexposed lower layers have greater water content so they are moresusceptive to being frozen during a procedure designed to freeze tissue,especially when an INA is used to facilitate the freeze. Permeability ofthe skin can also be increased by using microneedling whereby aplurality of microscopic holes are formed in the skin to create pathwaysfor absorption of a coupling media. Alternatively, sonophoresis can beused whereby ultrasound waves are used to stimulate micro-vibrationswithin the skin to increase the overall kinetic energy of moleculesmaking up the coupling media or topical agent to be delivered into theskin to increase absorption. Some preferred frequencies are 20-40 kHz,or more than 1 MHz. Other frequencies could be used. Alternatively,increased absorption can be achieved using iontophoresis techniques forincreasing absorption using, for example, electric fields to pushtopical agents into the skin. A permeability coefficient of couplingmedia for passing through tissue (e.g., epidermal tissue) can beincreased at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or95% to achieve desired absorption rates using any one or more of theabove described techniques. Other techniques can be used to facilitatedelivery of coupling media or substances. Different testing techniques(e.g., static cell techniques, flow-through diffusion techniques, etc.),algorithms, and modeling can be used to determine the permeabilitycoefficient and can be used to determine flux, including a steady stateflux.

At block 154, coupling media can be applied to the skin. The couplingmedia can include, without limitation, water, hydrogels,cryoprotectants, emulsions, combinations thereof, or the like beforepreparing the treatment. Applying the coupling media may includeplacing, spraying, coating, or rubbing a liquid, gel, or sheet couplingmedia onto the skin using an instrument including, for example, a brush,a spatula, a spray bottle or a syringe, or by hand (e.g., an operator'sgloved hand).

The coupling media can include one or more temperature depressants,INAs, etc. The temperature depressants can include, without limitation,polypropylene glycol (PPG), polyethylene glycol (PEG), propylene glycol,ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), or other glycols.The temperature depressants may also include ethanol, propanol,isopropanol, butanol, and/or other suitable alcohol compounds that maylower the freezing point of a solution (e.g., body fluid) to about 0° C.to −40° C., and more preferably to about −10° C. to −16° C. Certaintemperature depressants (e.g., PPG, PEG, etc.) may also be used toimprove smoothness and to provide lubrication. Additionally oralternatively, the coupling media can include one or more thickeningagents, pH buffers, humectants, surfactants, and/or additives.

At block 156, the subject's skin can be cooled. An applicator can beapplied to the treatment site to place the applicator in thermal contactwith target tissue. Tissue can be supercooled and then frozen, to limitor prevent unwanted side effects. The surface of a human subject's skincan be cooled to a temperature no lower than −40° C. to avoid unwantedskin damage. The surface of the skin can be heated to bring shallownon-targeted tissue out of the supercooled state while the deepertargeted region remains in the supercooled state.

At block 158, the supercooled targeted region can be nucleated toproduce freezing that can destroy or damage targeted cells, for example,due to crystallization of intracellular and/or extracellular fluids. Acatalyst for nucleation (e.g., mechanical perturbations, RF energy,alternating electric fields, etc.) can be provided following aprotective increase of a temperature of non-targeted epidermal layers.The mechanical perturbations can be vibrations, ultrasound pulses,and/or changes in pressure. Non-targeted layers of tissue can be warmedenough to avoid freezing upon nucleation of targeted tissue. Thetreatment systems disclosed herein can utilize applicators disclosedherein to perform such supercooling methods.

Some treatments include freezing dermal tissue more times than adjacentepidermal tissue. At block 156, dermal and epidermal tissue can becooled and frozen (block 158). The skin can be warmed by an applicator(which is at a temperature slightly below 0° C.) an amount sufficient toallow the dermal tissue to thaw due to internal body heat but not theepidermal tissue which is further removed from blood flow than is dermaltissue. After thawing the dermal tissue, block 158 can be repeated bychilling, for example, the skin to refreeze the dermal tissue while theepidermal tissue remains frozen. A desired level of damage to the dermaltissue can be achieved by repeatedly freezing and thawing the dermallayer, because the primary mechanism of damage during freezing is causedby ice crystal nucleation and growth.

Some treatments include a warm/thaw step 159 following the freezeevent(s) whereby the frozen and cooled tissue is rewarmed eitherpassively or actively by the applicator. After the warm/thaw step 159,the cooling 156 and freezing 158 steps can immediately be repeated, asshown by arrow 153, any number of times, such as 1, 2, 3, 4 or moretimes, preferably during the same patient treatment and optionallywithout moving the applicator. Alternatively, the cooling 156 andfreezing 158 steps can be repeated during the same patient treatment butafter the applicator has been moved to another treatment site and thenbrought back to the original treatment site, or during a separatepatient treatment session either later in the day of the first treatmentor the next day or several days later. Any number of repeat sessions canbe employed to achieve an overall desired level of treatment. Arrows 157and 155 show possibilities for the retreatment which can include arepeat of the skin preparation step 152 and/or a repeat of the applyingcoupling media step 154, as desired.

FIGS. 9A-9C show stages of a method for preparing a treatment site inaccordance with an embodiment of the disclosed technology. Generally,the subject's skin can be mechanically altered to facilitate absorptionof coupling media. For example, stripping elements can be applied to andremoved from the skin surface any number of times to remove an upperportion of the epidermis so as to expose lower layers of tissue. Thelower layers can have a relatively high water content and thus may bebetter able to absorb and uptake various agents, including water or oilbased coupling media, into the epidermis.

FIG. 9A is a cross-sectional view of a stripping element 200 applied tothe skin surface and overlaying a pore. The stripping element 200 can bean adhesive strip (e.g., adhesive tape) or another adhesive element thatcan be pulled off the skin to remove, for example, sebum, hairfollicles, or features from the pilosebaceous unit to expose the skinpore for uptake of applied substances. The stripping element 200 may bea single adhesive strip (e.g., piece of adhesive tape) that is cut tooverlay the entire treatment area. In other embodiments, multiplestripping elements 200 are applied to the treatment site. The adhesivecharacteristics of the stripping elements can be selected based on thedesired amount of mechanical alteration to the skin and desired patientcomfort.

FIG. 9B is a cross-sectional view of the stripping element 200 beingremoved from the skin to remove material 201 from a pore 203 to open orunclog a pore entrance 202. Additionally stripping elements can bereapplied any number of times to further unclog the pore 203 orotherwise prepare the treatment site.

FIG. 9C is a cross-sectional view of a treatment site after the pore 203has been cleared and a substance 205 has been applied. The substance 205can infuse the pore 203 and can be absorbed by the skin. Water can bepart of the substance, and a subsequent freeze event can cause the waterin the skin pore to freeze and cause additional tissue damage.

Skin can be mechanically stimulated before, during, and/or after anysteps in the method 140 (FIG. 5 ) or 150 (FIG. 8 ). Mechanicalstimulation can include, for example, stimulation or agitation bybrushing, rubbing, applying ultrasound, dermabrasion, or other meanswhich can clean the treatment site and/or cause the barrier of thestratum corneum (i.e., the outermost layer of the epidermis consistingof dead cells) to be temporarily reduced and/or increase movement (e.g.,turbulence) of the coupling media with respect to the skin. Withoutbeing bound by theory, it is believed that mechanical stimulation of theskin (e.g., agitation of, reduction of, or penetration of the stratumcorneum) can enhance the permeation of the coupling media into theunderlying epidermal layer, dermal layer, or another layer of tissue. Inone embodiment, the skin can be mechanically stimulated for about 20seconds to about 10 minutes. In another embodiment, mechanicalstimulation can be applied to the treatment site for about 20 seconds,about 40 seconds, about 1 minute, about 2 minutes, about 5 minutes orgreater than about 5 minutes. In some embodiments, mechanicalstimulation could be performed with, for example, a dermal agitationbrush, a brush having rotating bristles, or the like. Brushing orrubbing the skin can include, in some embodiments, moving across theskin at the treatment site in a circular or back-and-forth motion or, inother embodiments, in linear strokes, for increasing the skinpermeability for the substance. The permeation rate can be increased ordecreased to achieve a desired amount of absorption.

Different techniques can be used to evaluate the permeability of theskin before and/or after performing the stripping process. In oneprocedure, the coupling agent can be applied to the treatment site andthen cells at the treatment site can be progressively removed byrepeatedly applying the stripping element or by applying a series ofstripping elements. The stripping elements and treatment site can beevaluated to determine the volume of the coupling media absorbed by theskin.

D. Substances for Treatments

Because ice crystals can be reliably generated to trigger on-commandfreeze events, a substance can be used to improve thermal couplingbetween a skin surface and a cooling applicator. In some embodiments,the substance is an aqueous solution coupling agent, which contains acryoprotectant agent. Further substances can contain water and a mediumfor promoting an on-demand reliable creation of an ice crystal wheninitiation of freezing in the treatment is desired. Liquid water hasclusters of molecules that are undergoing constant collisions with othermolecules and clusters, sometimes breaking apart and sometimes formingnew clusters. When water is being cooled, as the temperature drops andthe thermal movement of water molecules decreases, the tendency of watermolecules to aggregate becomes stronger and the likelihood that acritically large cluster of molecules will form increases rapidly. Icenucleation is catalyzed upon formation of a critically large cluster ofmolecules. Consequently, initiation of freeze or ice nucleation in asample of water (or a coupling agent) takes place from a nucleus with anice-like structure. The nucleus can promote the organization of watermolecules into an ice crystal lattice.

Water and aqueous coupling agents have a natural tendency to cool to atemperature significantly below their equilibrium freezing point beforeice nucleation; that is, they have a tendency to supercool. There aretwo modes of ice nucleation of water: homogenous and heterogeneous. Whena critically large nucleus is formed by spontaneous aggregation of thewater molecules themselves, the nucleation is referred to as“homogeneous.” For a macroscopic quantity of water, the size of acluster needed for ice nucleation is often about 25 molecules. Theradius of the cluster can be about 3 molecules. The critical radius thatcoincides with this size gives a temperature of −41° C., which is calledthe homogeneous nucleation temperature for water. Accordingly, thehomogeneous nucleation temperature for water is the minimum temperaturethat pure water can be cooled to before freezing occurs spontaneously.

When aggregation of water molecules is catalyzed by an external source,the nucleation is referred to as “heterogeneous.” The cause of externalnucleation can be the introduction of ice crystals or another externalsubstance into the supercooled sample. For example, crystallization canbe triggered by the physical introduction of a nucleation initiator(e.g., a seed crystal or nucleus) around which a crystal structure canform to create a solid.

The substances can be hydrogels, liposomes, or emulsions, such asoil-in-water (O/W) emulsions, water-in-oil (W/O) emulsions, oil-in-oil(O/O) emulsions, or nano-emulsions, and can provide homogeneous orheterogeneous nucleation.

1. Nucleation Initiators

An INA can be a substance that promotes the formation of a seed crystal(or initial cluster), thus catalyzing a heterogeneous ice nucleation.When INAs are used, water freezing takes place at a temperature higherthan would be required in the case of a homogeneous nucleation, and thelargest biological ice nucleators may trigger freezing at −1° C. to −5°C. or other lower temperatures, long before a spontaneous water freezewould normally otherwise occur. Spontaneous water freezes can variablyoccur at −10° C., −15° C., −20° C., or −25° C. or lower, and the timingof the spontaneous freezes is very unpredictable. Example INAs includebiogenic-derived proteins, materials derived from a Gram-negativeepiphytic bacteria, and/or materials belonging to the genus Pseudomonas,Erwinia, or Xanthomonas. For example, INAs may be inorganic- ororganic-derived substances that promote heterogeneous ice nucleation.Embodiments of the present technology can include methods of producingcontrolled and predictable freezes of the skin and subcutaneous tissueusing INAs.

In general, INAs can promote the formation of ice crystals in water-likesubstances at a specific temperature, such as generally a few degreesbelow 0° C. INAs can be used synergistically with a selected temperaturetreatment protocol to control the onset and extent of a freeze eventduring cryotherapy and can be used to promote in-vivo freezing at highertemperatures than a natural homogeneous nucleation freezing point ofskin tissue. An aspect of some embodiments of the present technologyrelates to methods of producing a controlled freeze of the skin andsubcutaneous tissue using INAs. Cooling methods using INAs permit thetriggering of ice nucleation at specific temperatures, such astemperatures close to 0° C. Thus, INAs may provide an advantage intherapies that require freezing and variable treatment temperatures withdesired therapeutic treatment/safe temperature ranges and that create aprecise and controllable extent of skin and tissue damage from a freezeevent.

Various Gram-negative epiphytic bacteria have been known to produceINAs. These belong to the genera Pseudomonas, Erwinia and Xanthomonas,among others. One of the highest level of ice nucleation activators isan ice nucleation protein (INP) from some ice-nucleating bacteria.Protein molecules and materials located on the outer membrane of thesebacteria are responsible for the ice nucleation. Cells can also be lysedor otherwise produce pieces of cellular material (e.g., membranes) inwhich such INAs are found or trapped, for example, Pseudomonas Syringae.

One commercially accessible INA is SNOMAX® available from Snomax LLC,Englewood, Colo., which is derived from the bacterium PseudomonasSyringae (freeze-dried protein powder). This protein initiates afreezing process by serving as an ice nucleator and raises thepredictable freezing temperature of water to about −3° C. SNOMAX® isused widely for snowmaking and is safe for human use and non-pathogenic.SNOMAX® can be a powder that exhibits 10¹² to 10¹³ ice nuclei per gramat temperatures less than about −4° C. Coupling agents prepared withSNOMAX® or other substances derived from the bacterium PseudomonasSyringae can have enough INAs to produce reliable ice nucleation atdesired temperatures. Bacteria/cell concentration has a direct effect onthe nucleation temperature of water. INAs can be used in standard powderform, and can be used as ice nucleators with or without added water. Insome embodiments, INAs can be fractionally delivered to skin, such as bymicroneedles (e.g., an array of microneedles). Biocompatible INAs can beinvasively delivered using needles, such as intradermal needles.Additionally or alternatively, INAs can also be used with non-contactcooling devices, such as cooling/freezing sprays.

INAs can be used in cooling protocols to cause ice nucleation attemperatures about −2° C., −3° C., or −4° C. At these temperatures,damage to epidermal tissue can be significantly less than damagetypically produced at lower freezing temperatures. The temperature forice nucleation can be selected to be high enough to avoid significantskin pigmentation changes associated with freeze events.

A non-invasive applicator (e.g., applicator 104 of FIGS. 2 and 3 ) canbe used to control skin cooling and can include one or more temperaturesensors. Temperature sensors (e.g., element 167 in FIG. 2 ) can beembedded along the treatment surface of the applicator and can be usedas part of a temperature control system. The temperature control systemcan include one or more feedback control algorithms to control theapplicator based on a predetermined set of temperature values over oneor more predetermined periods of time, and have predetermined rates ofchange when transitioning from one temperature to another temperature,and so on. Different feedback control algorithms can be used to treattissue using different treatment temperature protocols (and createdifferent temperature treatment cycles) by varying cooling/thawingrates, predetermining therapeutic treatment temperatures, and/orselecting treatment durations. The methods described herein can involveusing both INAs to control freezing and varying treatment temperatureprotocols and profiles.

2. Hydrogel Materials

An aspect of the present technology relates to methods of using hydrogelsubstances with freezing point depressants (cryoprotectants) and/or INAsfor creating a controlled “on command” predictable freeze. Hydrogelsubstances are a class of crosslinked polymers that, due to theirhydrophilic nature, can absorb large quantities of water. Hydrogelsubstances can have a suitable water content for controlling freezing,including controlling ice nucleation, ice crystallization, freezepropagation, or the like. Integral parts of the hydrogel synthesisinclude a monomer, an initiator, and a crosslinker. Hydrogel propertiescan be modulated by varying their synthetic factors, such as reactiontemperature, monomer type, monomer crosslinker, crosslinker-to-monomerratio, monomer concentration, and type and amount of initiator. Thecomposition of hydrogels can be selected for a specific application byselecting proper starting materials and processing techniques.

Hydrogels can be mixed with one or more freezing point depressants andcan be engineered to have desired melting/freezing temperatures (e.g.,optimum melting temperatures). The freezing point depressants caninoculate tissue. Additionally or alternatively, hydrogels can becombined with INAs that have a set activation temperature to make thehydrogels able to freeze consistently at predetermined temperatureranges (or a specific temperature) different from those associated withhydrogels without ice nucleating agents. The combination of hydrogels,freezing point depressants, and/or INAs can result in a controllablefreeze at desired temperatures, such as −3° C., −2° C., −1° C., or othertemperatures. Temperatures close to 0° C. can be less damaging toepidermal tissue and are well suited for less aggressive temperaturefreezing protocols, so temperatures can be selected to protect one ormore upper layers of the skin to eliminate or minimize any substantialdiscoloration side effects associated with freezing skin treatments andto eliminate any permanent adverse events.

The water accommodated by the hydrogel structure can be classified infour types: free, interstitial, bound, and semi-bound water. Free wateris located in the outermost layer and can be easily removed fromhydrogels under mild conditions. Interstitial water is not attached tothe hydrogel network but is physically trapped between the hydratedpolymer chains. Bound water is directly attached to the polymer chainthrough hydration of the functional groups or ions. The bound waterremains as an integral part of the hydrogel structure and can beseparated only at very high temperatures. Semi-bound water hasintermediate properties of bound water and free water. The free andinterstitial water can be removed from the hydrogels by centrifugationand mechanical compression.

Controlled freeze techniques can take advantage of the water compositionof hydrogels. Hydrogels can be designed to have a specific freezingpoint or specific freezing temperature range by having a specific ratioof water-monomer-crosslinker content. Cryoprotectant additives, such asglycols (e.g., PG) or other substances, can be used as well to lowertheir freezing point.

A hydrogel can act as an initiator of a predictable freeze event. As thehydrogel freezes, the hydrogel provides “initial seeds” or crystal sitesto inoculate tissue and thus catalyze a controlled predictable freeze ata specific temperature in the skin. In some embodiments, a predictablefreeze event can be freezing of tissue that occurs at least 90%, 95%, or98% of the time when freezing is desired. A predictable controlledfreeze event in a hydrogel can also be achieved by precooling thehydrogel to a temperature below its melting point. The freeze event canbe initiated by injecting a nucleation initiator (e.g., ice/waterslurry) into the hydrogel to create freezing that reaches the surface ofthe hydrogel adjacent the patient, which causes freezing of thesubject's skin. In other procedures, ultrasound or other nucleationenergy can be used to produce a freeze event in the hydrogel. Accordingto one embodiment, additives (e.g., cryoprotectants and/or INAs) can beembedded in isolated layers within an interior of the hydrogel so thatthese substances are not on an exterior surface of the hydrogel sheet orhydrogel pad and hence do not come in direct contact with skin or othertissue being treated. Encapsulating these substances within the hydrogelobviates the need to choose INA substances that have been tested andvalidated to be safe when in contact with skin or tissue. Predictablehydrogel freezes can be enhanced by additives, such as INAs.

FIG. 10A shows a patterned hydrogel in accordance with an embodiment ofthe disclosed technology. FIGS. 10B and 10C are plots of PGconcentration (% PG) versus length for patterned hydrogels. Hydrogelscan include a dispersion medium of water and volumes of nucleationinhibitors (e.g., particles or columns of water/PG emulsion). FIG. 10Ashows isolated volumes of nucleation inhibitors spaced apart within avolume of water. In some embodiments, a hydrogel layer or sheet cancontain columns of a nucleation inhibiting emulsion separated by avolume of water with substantially no PG. The water surrounding thecolumns can serve as ice nucleation sites. The inner enclosures (e.g.,encapsulants) can inhibit or prevent diffusion of the water/PG emulsion.The composition of the inner enclosure can be selected based on thecomposition of the enclosed substance, and the pattern, number, andsizes of the localized nucleation inhibiting volumes can be selectedbased on the hydrogel characteristics.

FIGS. 10B and 10C show embodiments with propylene glycol (PG) locatedthroughout the hydrogel, with the concentration of the PG varying alongthe length and width of a layer or a sheet of hydrogel. Regions of thehydrogel having the lowest concentration of PG have the highestmelting/freezing point and can thus function as ice nucleating regions.Isolated freezing zones can be formed in the skin adjacent to those icenucleating regions. Other types of cryoprotectant agents or componentscan replace PG. For example, PG can be replaced or combined with PPG,PEG, DMSO, or the like.

A further embodiment is a hydrogel that contains an INA placed uniformlythroughout areas where it is desired to seed freeze propagation.Further, the INA can be dispersed exclusively within interior portionsof a volume of hydrogel. For example, the INA can be within a hydrogelsheet so that the INA does not extend to a surface of the sheet, therebypreventing contact between the INA and the skin. The INA can seed afreeze event in an interior region of the hydrogel, and the freeze eventcan rapidly propagate to an outer surface of the hydrogel, which in turncontacts the skin and causes a freeze event in the skin.

FIGS. 11A and 11B are side views of hydrogels with ice nucleatingregions. FIG. 11A shows a hydrogel material with an interior icenucleating region between upper and lower ice nucleating inhibitingregions. The ice nucleating region can comprise an INA and can have arelatively low concentration of temperature depressant, if any. In oneembodiment, the ice nucleating region can be substantially free oftemperature depressants and can comprise water and ice nucleatingfeatures (e.g., INAs, ice nucleating particles, or the like). The icenucleating region can be a layer, either a continuous layer or as spotson an interior layer so as to be totally embedded within the hydrogel.

The ice nucleating inhibiting regions can have a melting/freezing pointlower than the ice nucleating region and can include volumes oftemperature depressants, such as evenly or unevenly spaced apart volumesof PG. The pattern, number, and composition of freeze inhibitingfeatures can be selected based on the desired nucleation inhibitingcharacteristics.

FIG. 11B shows a multilayer hydrogel material with outer ice nucleatinginhibiting layers and an inner ice nucleating layer. The outer icenucleating inhibiting layers can include temperature depressants, andthe inner layer can comprise a high centration of INA (e.g., mostly INAby volume). For example, the outer ice nucleating inhibiting layers canbe a layer of a PG solution or a layer with a dense array of PG volumes,and the inner layer can comprise mostly or entirely water. A freezeevent can be initiated in the ice nucleating layer and then spreadthrough the outer ice nucleating inhibiting layers.

The hydrogel can be sticky on both a patient side and an applicatorside. Sticky upper and lower surfaces can help maintain contact with thesubject's skin and applicator and, in some embodiments, help to minimizeor limit movement of the hydrogel during treatment. A liner can be usedto prevent contamination of the hydrogel. A side of the hydrogel thatcontacts a liner can be sticky. In some embodiments, the hydrogel can bea sheet with a uniform or variable thickness with adhesive applied toone or more of its outer surfaces.

Hydrogels can be used in the methods discussed in connection with FIGS.5, 8, and 9A-9C. For example, at block 142 in FIG. 5 , a hydrogel can beapplied to the subject's skin and can include an INA capable of formingice crystals in the presence of water. The INA can be encapsulatedwithin a polymer structure of the hydrogel such that the INA does notcome in direct contact with the skin as discussed in connection withFIGS. 11A and 11B. The hydrogel and skin can be cooled to arrive at asuitable cooling temperature for freezing the skin. The hydrogel caninclude a freezing point depressant such that a first melting/freezingtemperature of the hydrogel is lower than a second melting/freezingtemperature of fluid in the skin.

At block 144 in FIG. 5 , skin can be cooled to a temperature above thefirst freezing temperature and below the second freezing temperature soas to supercool the skin, and after a predetermined amount ofsupercooling has occurred, the skin is frozen at block 146. Thetemperature of the epidermis can be raised above the first temperatureprior to freezing the dermis.

Referring to the method 150 in FIG. 8 , a hydrogel substance comprisinga crosslinked polymer and an INA can be applied at block 154. The INAcan be embedded within a polymer structure to prevent direct contactbetween the INA and the skin. In some embodiments, a sheet of hydrogelcan be applied to the subject's skin. Alternatively, hydrogel can beinjected into the skin. Other techniques can be used to apply hydrogels,which can be creams, gels, etc. At block 156, the skin is cooled. Atblock 158, the freeze event can be initiated using one or more icecrystals. In other embodiments, energy is used to break the structurescontaining INAs to release a sufficient amount of the INA to produce afreeze event.

3. Liposomes

Liposomal transport of substances into tissue can be used to deliversubstances to specific tissue in a more effective manner than by justapplying the substances to a surface of the skin. Because a liposome islipophilic, it can be absorbed at least into the stratum corneum and canthen release a substance within the liposome at a specific location ordepth in the subject's tissue. Liposomes can trap water in significant“buckets” that enhance the water content of skin when the liposomebreaks down, and make freeze protection more predictable when used withsignificant amounts of cryoprotectant in the water in the liposome.Liposome skin hydration can be more effective than directly applyingwater to a skin surface since the stratum corneum is normallyhydrophobic.

A topically applied liposome can enhance thermal contact between theapplicator/skin and can provide controlled delivery of agents (e.g.,cryoprotectant, INAs, etc.), and the liposomes can penetrate the stratumcorneum better than either water or water mixed with a cryoprotectant.Additionally, liposomes can deliver different agents to differentlocations, thus allowing direct transfer of agents to specific targetedcells. In one embodiment, the liposome contains a cryoprotectant (e.g.,propylene glycol) and can break down to release the cryoprotectant. Inanother embodiment, the liposome selectively releases an INA to providecontrolled freezing capability through specific tissue.

According to embodiments where freezing is desired, substances (e.g.,INAs, cryoprotectant, etc.) can be incorporated into liposomes such thatthe liposomes can controllably release the substances into the skin.Specifically, liposomes can be formulated to maintain their structurewhen penetrating the skin to minimize, limit, or substantially preventrelease of substances. When enough liposomes accumulate in a certaindesired tissue or layer of the skin, an “on-command” breakdown of theliposomes can be initiated to trigger a burst release of the embeddedagents. In some embodiments, the liposome can contain an INA forinitiating freeze events. Triggering methods for breaking down liposomesinclude using temperature (e.g., temperature cycling), ultrasound, or acleansing agent to disrupt or break lipid encapsulation of theliposomes. An applicator can include heaters for heating the treatmentsite to cause release of the agents, can include transducers fordelivering mechanical energy in the form of ultrasound waves, or caninclude other elements for disrupting liposomes to perform the methodsdiscussed in connection with FIGS. 5 and 8 .

Liposomes can have compositions selected based on, for example, a rateof agent release, stability, and/or other desired characteristics. Insome embodiments, the rate of agent release can be increased by applyingenergy, such as ultrasound, heat, or other energy suitable for breakingdown lipids that entrap agents. For example, a media can include firstliposomes for delivering cryoprotectants to the epidermis and secondliposomes for delivering INAs to the dermis. Once the first liposomesare absorbed by the epidermis, they can release the cryoprotectant toprotect the epidermis. After the second liposomes have passed throughthe epidermis and been absorbed by the dermis, they release the INA intothe dermal tissue. Upon cooling the treatment site to a temperaturebelow a melting/freezing point of the skin, the INA can cause apredictable freeze in the dermal tissue. Accordingly, each agent can bedelivered to specific locations using liposomes. Liposomal medias can beused before, during, and/or after a treatment session. In someprocedures, a topical media is applied to the skin surface to delivercryoprotectant to shallow tissue before cooling. Another media (e.g.,media with an INA) can be injected into deeper tissue once the tissue iscooled and is ready for freezing.

4. Emulsions

Emulsions are a class of disperse systems comprising two immiscibleliquids and can contain liquid droplets, which comprise the dispersephase, dispersed in a liquid medium, which is the continuous phase.Emulsions can be oil-in-water (O/W) emulsions, water-in-oil (W/O)emulsions, oil-in-oil (O/O) emulsions, or nano-emulsions. Nano-emulsionsare desirable since they can penetrate the epidermis and dermis alonghair follicle apertures and skin pore apertures. FIG. 12A shows anemulsifier or surfactant with a hydrophilic head and a hydrophobic tail.FIG. 12B shows an agent (e.g., oil-based agent) entrapped by theemulsifiers to separate the agent and water. FIGS. 13A and 13B showoil-in-water and water-in-oil emulsions. Referring to FIG. 13A, theemulsion includes oil droplets that can comprise the same or differentagents. In a single-agent emulsion, each droplet can comprise the sameagent. In a multi-agent emulsion, different agents (e.g., dispersedmediums) can be evenly or unevenly dispersed in the dispersion medium.The dispersed medium can include droplets containing one or morecryoprotectants, INAs, analgesics, agents, or the like.

FIG. 14 is a table with melting/freezing point temperatures for fats.The fats can be natural oils suitable for O/W emulsions and can haverelatively high melting points. For example, fats can havemelting/freeze points above 0° C. and can be used in emulsions.

E. Tests and Methods of Treatment

Ex-vivo bench tests with skin using treatment cycles show theunpredictability of supercooling skin and precise control freezing withINAs. In one test, a thermocouple was placed between a coupling layer(coupling media) and skin to detect freezing. A coupling layer of onlywater was tested to confirm there is no freezing of tissue without anINA. Thermocouple temperature data of five tests, including twowater-only tests (where no freezes occurred) and three tests using anINA (where three separate freezes occurred), were conducted to show theeffects of INAs and the feasibility of the controlled freeze concept.

FIGS. 15A and 15B show the results of the tests: with INAs the skin wasconsistently predictably inoculated and frozen three separate times,whereas without INAs the skin tended to merely supercool and not freezethe two separate times an INA was not used. FIG. 15A shows an exampletreatment profile designed to supercool at −2° C. for 3 minutes and totrigger freezing at −5° C. FIG. 15B shows three tests with INAs andfreeze events occurring shortly after 200 seconds and the associatedtemperature increase caused by the heat of fusion, which causes themeasured temperature to increase from about −1° C. to about 0° C. Anon-invasive surface cooling apparatus was used to perform the testsdiscussed in connection with FIGS. 15A and 15B, and the INA was aSNOMAX®/water solution.

FIG. 16 shows an example treatment cycle temperature profile and atemperature response of the thermal sensor at the applicatorsurface-tissue interface. A target temperature of atemperature-controlled surface of the applicator (shown in dashed line)can be lowered at a predetermined rate and then held constant at apreset value. One or more sensors can monitor the temperature at thecontact interface. Output from the sensors can be used to preciselycontrol freeze execution in order to maximize cellular changes with atherapeutic purpose while inhibiting, limiting, or minimizing adversetreatment side effects. It may also be desirable to control the extentof skin freezing down to the epidermal-dermal layer, dermal-subcutaneouslayer, or other specific depths. A desired freezing extent can beachieved based on knowledge of the freezing point temperature of thebulk tissue. In freeze events, tissue goes through ice nucleation andgrowth (exothermic phase change). An exothermic phase change is when thesystem releases heat to the surroundings (e.g., changing from a liquidto a solid) during the phase change. Freezing is an exothermic event,releasing heat for a very short time period, and this release of heatcan be a reliable indicator of skin freezing. Thermal sensors can beused to detect the heat released by the phase change associated with afreeze event. When tissue is at a supercooled steady state, a thermalsensor (e.g., element 167 in FIG. 2 ) at the applicator surface-tissuecontact location can detect that the temperature is stable, without anysubstantial sudden temperature changes or peaks. When the supercooledtissue freezes, the released heat is captured by the sensor as a suddenincrease of temperature. FIG. 15B shows temperature increases in tests2, 3, and 5 corresponding to the released heat.

FIG. 17 shows the set temperature of the applicator surface (shown indashed line) lowered at a constant rate and then held at a generallyconstant value. The temperature at the applicator-tissue interface isdetected by the thermal sensor and shown in solid line). After targetedtissue has been supercooled, a freeze event can be initiated. Thetemperature sensor can detect the temperature increase associated withthe heat of fusion, and the detected increase in temperature can beidentified as a change in phase. Various changes in temperature can beused to monitor tissue and detect phase changes. Some methods caninclude supercooling and using freezing set points to intentionallytreat tissues at subzero temperatures. The tissue temperature can belowered at a desired time during treatment to control onset of freezing.Thaw cycles can be included in any treatment cycle to warm tissue at adesired rate for protecting or enhancing tissue injury.

A treatment cycle can be determined by selecting supercoolingparameters, freeze parameters, and thaw parameters. The supercoolingparameters can include cooling profiles, target temperatures, and/ortime periods. A cooling profile can include a cooling rate for ramp-downto reach the supercool temperature. The target tissue can be kept at asubzero target supercool temperature for the supercool time periodwithout phase change.

Freeze parameters can include cooling profiles for keeping the tissue inthe frozen state and/or time periods for keeping the tissue frozen. Thefreeze parameters can be selected to increase or decrease thermalinjury. After completion of the freeze time period, cooled tissue can bewarmed using a thawing cycle.

Supercooling parameters, freeze parameters, and/or thaw parameters canbe obtained experimentally ex-vivo and/or in-vivo. For example, in-vivohuman tests have shown that skin can be supercooled to subzerotemperatures, for example, as low as −20° C., without phase change. Whenthe temperature is lowered far enough, the tissue will freeze. It hasbeen experimentally established that human skin tissue will oftenspontaneously freeze at around −25° C. The temperature of spontaneousfreezing depends on the characteristics of the tissue, such as watercontent of tissue, cellular structure, etc.

FIG. 18 shows an exemplary treatment cycle for controlled supercoolingand for controlled freezing. Temperature gradients within tissue duringcooling protocols can be estimated by biothermal heat transfer modeling,experimental tests, or a combination of both. Heat transfer modeling canbe used to predict the temperature gradients within tissue versus timeduring a treatment cycle. The volume of tissue, tissue depth, and otherinformation about tissue at subzero temperatures can be calculated. Forexample, a flat applicator can cool skin to supercool targeted tissuebetween 0° C. to −20° C., 0° C. to −12° C., 0° C. to −10° C., 0° C. to−8° C. or other suitable temperature ranges to cool targeted tissue to atemperature equal to or lower than about −20° C., −15° C., −13° C., −12°C., −11° C., −10° C., −8° C., −6° C., −5° C., −4° C., −3° C., −2° C.,−1° C., or 0° C. The target tissue can reach a general steady state atwhich the subject's body heat offsets continued heat withdrawal from theskin surface. The temperature-controlled surface of the applicator canbe kept at a suitable temperature lower than the desired bulktemperature of the targeted tissue for the supercooling period. Tofreeze the supercooled tissue, a temperature of the applicator can befurther lowered to ramp down to, for example, a melting/freezing pointof a medium on the skin, a freezing point of an agent in the skin, or afreezing point of the skin itself.

FIGS. 19A-19E are cross-sectional views of a cooling applicator appliedto a treatment site and thermal modeling. FIG. 19A shows the treatmentsite at the start of a supercooling cooling cycle when shallow tissuebegins to be supercooled. FIGS. 19B-19E show the amount of supercooledtissue increasing over time until the entire epidermis/dermis underlyinga cooling plate is supercooled, while the subcutaneous layer is notsupercooled. (Skin tissue that is supercooled is gray, and thenon-supercooled tissue is black.) The supercooled tissue can be at atemperature within a range of about 0° C. to −20° C. The model forcooling skin is based on a flat plate applicator (with a treatmentprofile that ramps down at 1° C./s and a holding step at a targettemperature of −20° C.) to predict that skin with an epidermal layer of0.1 mm and a dermal layer of 2 mm, which are above a subcutaneous layerof 1.0 cm (10 mm), can be supercooled completely within 90 seconds. Tofreeze the whole underlying region of the epidermal/dermal layers, skinfreeze can be triggered to occur at or after 90 seconds of supercooling.To freeze only a portion of underlying epidermis/dermis, skin freeze canbe triggered to occur before 90 seconds (for example, after 45, 60, or75 seconds of supercooling). The onset of the freeze event may bedelayed for a short period of time (e.g., a few seconds) after thefreezing temperature is reached due to the time it takes to cool thetissue to a lower set temperature and the time it takes for an icenucleation event to occur, so some additional time may need to be addedfrom a time a temperature dive is initiated until a thaw event is begun,to result in a desired amount of time the tissue is in a partiallyfrozen state. For skin, approximately 15 seconds, 20 seconds, or 30seconds can be average delay times from the beginning of a temperaturedive until the freeze event has occurred.

When a freeze event is initiated, the entire dermis and epidermisunderlying the applicator may not be completely frozen. This is becauseeven at steady state (e.g., when heat extraction by the applicatorbalances warming from subdermal tissue and blood flow) the bulktemperature of the tissue is not cold enough to absorb all the heat offusion from the freeze event so as to achieve a 100% tissue freeze. Asthe heat of fusion is released during the freeze event, the bulktemperature of the tissue rises to a level close to, for example, 0° C.such that additional freezing ceases (absent additional significant heatextraction by the applicator) and the skin is only partially frozen whenequilibrium is established. The temperature of the cooling plate can beadjusted to compensate for heat of fusion or other natural heatingassociated with the subject's body. Without being bound by theory, it isbelieved that the skin would need to be supercooled to a temperaturearound −70° C. for the skin to totally freeze and remain complete frozenduring a freeze event, but such a low supercool temperature is highlyundesirable because severe adverse events, particularly to theepidermis, would result.

FIGS. 20A-20F illustrate stages of one method of freezing tissue withoutsupercooling. Generally, an applicator can be applied to a treatmentsite after applying a coupling agent to the applicator and/or treatmentsite. The applicator can freeze a region of the skin while limiting oravoiding supercooling, as discussed below.

FIG. 20A shows a coupling media 209 (e.g., water, coupling gel, etc.)located along a temperature-controlled surface 211 of the applicator.The layer of coupling media can be formed by applying water to thetemperature-controlled surface 211, which can be cooled to a temperatureless than about −20° C., −15° C., −10° C., or another suitabletemperature for freezing most of or all the coupling media 209. Thetemperature-controlled surface 211 can then be warmed to a highertemperature (e.g., −3° C., −2° C., or −1° C.) suitable for applying theapplicator to the treatment site.

FIG. 20B shows a frozen upper layer 215 and a liquid lower layer 217 ofthe coupling media after the applicator has been applied to thetreatment site 213. The warm skin can contact the liquid layer 217 gelwhile the layer of the frozen coupling media contacts thetemperature-controlled surface 211 and remains frozen. Thetemperature-controlled surface 211 can be held at a temperature suitablefor maintaining the frozen upper layer 215 and the liquid lower layer217.

FIG. 20C shows the temperature-controlled surface 211 held at lowtemperature (e.g., −1° C., −2° C., −3° C., etc.) for a predeterminedperiod of time to cool the skin to a temperature near itsmelting/freezing point. During this holding period, a portion of thecoupling media which is frozen may slightly increase in volume, therebyincreasing the thickness of the layer 215. The temperature of thetemperature-controlled surface can be lowered or raised to increase ordecrease the thickness of the frozen layer 215 while maintaining aliquid layer 217. In some procedures, the temperature-controlled surface211 can be kept at a temperature within a temperature range of about −2°C. to about −12° C., about −4° C. to about −10° C., or other suitabletemperature ranges. For example, the temperature-controlled surface 211can be kept at a temperature of about −6° C., −8° C., or −10° C. As thetemperature is reduced and/or held a lowered temperature, the freezingfront of the coupling media moves towards the skin until substantiallyall or the entire layer of coupling media/gel freezes.

FIG. 20D shows the entire layer of coupling media 209 in a frozen state.The surface of the skin contacting the coupling media 209 can be loweredto its freezing point. Due to the skin's intimate contact with icecrystals in the frozen coupling gel 209, the skin will progressivelyfreeze rather than supercool.

FIG. 20E shows a freezing front 221 and a frozen volume of tissue. Thefreezing front 221 can move deeper into the subject until a desiredvolume of tissue is frozen.

FIG. 20F shows temperature-controlled surface 211 held at a temperatureof about −8° C. for 2 minutes to produce the enlarged volume of frozentissue. The frozen volume of tissue can stop increasing and becomeconstant when the steady state is established. In facial treatments,skin can be frozen without affecting underlying muscles and subcutaneoustissue. In some treatments, the applicator can freeze skin withoutaffecting subcutaneous tissue to limit or avoid changing skin contoursat the treatment site. In other treatments, subcutaneous tissue can befrozen to, for example, inhibit, disrupt, or reduce subcutaneouslipid-rich cells to contour the treatment site. For example, theapplicator can treat acne and can also contour or not contour tissue ina single session.

The temperature of the temperature-controlled surface 211 can beincreased to 18° C., 20° C., or 22° C. at a rate of 1° C./s, 2° C./s, or3° C./s. This will quickly thaw the tissue to minimize or limit furtherdamage to cells. For example, the skin can be cooled at a rate of 0.25°C./s, held at a target temperature of −8° C. for 2 minutes, and thenthawed at a rate of 2° C./s. Other cooling rates, target temperatures,and thaw rates can be selected based on the desired level of freezing,thermal injury, etc.

Targeted tissue can be frozen more times than non-targeted tissue.Repetitive freeze-thaw cycles effectively damage or kill tissue because,aside from suffering multiple cycles of deleterious solution effect andmechanical ice crystal damage, cell membrane integrity will becompromised after the first freeze-thaw cycle, making it a lesseffective barrier for freeze propagation in subsequent freeze-thawcycles, and cells are much more susceptible to lethal intracellular iceformation in the subsequent freeze-thaw cycles. In some embodiments,targeted tissue can be frozen multiple times in a single treatmentsession while freezing non-targeted tissue, such as the epidermis, onlyone time. Additionally, targeted tissue can be frozen multiple timeswithout supercooling any tissue. In some procedures, the dermis isrepeatedly frozen to injure or destroy target glands without repeatedlyfreezing the epidermis.

FIG. 21 is a plot of temperature versus time for freezing the skinmultiple times in accordance with an embodiment of the disclosedtechnology. After a layer of frozen coupling media is created at −15°C., the temperature-controlled surface of the applicator is warmed to−2° C. to cool the skin using techniques discussed in connection withFIGS. 20A-20F. Thereafter, the freeze event happens while theskin-applicator interface is maintained at a temperature of about −2° C.(indicated by the “*”). The temperature-controlled surface is thencooled to −10° C., and the freeze event is held at −10° C. for a periodof time (e.g., 1 minute, 2 minutes, etc.). FIG. 21 shows a 1 minuteholding period. Thereafter, the temperature of the applicator is raisedto −1.5° C. for about 35 seconds. At this temperature, the epidermis,which is in contact with the coupling media, can remain frozen. Thedermis thaws due to internal body heat and, in particular, heat fromblood flow which perfuses the dermis. Thereafter, the applicator isre-cooled to −10° C. or another suitable temperature for refreezing thedermis. The second freeze event can be held for a period of time, suchas 1 minute, 2 minutes, etc. If desired, the dermis can be thawed againby warming the applicator to −1.5° C. and again refrozen at −10° C.while keeping the epidermis in a frozen non-thawed state. The thawtemperatures, warming rates, cooling rates, duration of freeze events,and refreeze temperatures can be selected based on the desired number ofrefreezes and severity of thermal injuries.

Because the epidermis is never thawed using this treatment protocol, alarger freezing rate will have a much less damaging effect on theepidermis. In second or subsequent freeze-thaw cycles, a much largerfreezing rate can be used to transition from a thaw temperature (e.g.,−1.5° C., −1° C., 0.5° C., etc.) to a refreeze temperature (e.g., −8°C., −10° C., −12° C.), and this may further increase the probability ofintracellular ice formation in the dermis, as further explained below.

The repeated freezing approach allows for complete control over most orall or some of the variables that govern post-thaw tissue viability.These variables include, without limitation, skin freezing rate, targettemperature, duration of freeze, and warming rate. The skin freezingrate is not as controllable using other approaches when skin issubstantially supercooled because a macroscopic freeze event happensalmost instantaneously (over a period of a few seconds) when skin isnucleated or inoculated with ice when the skin is in a supercooledstate. Without being bound by theory, it is believed that the freezingrate is important because in a procedure in which extracellular spaceice formation is triggered at −2° C., if the tissue is then slowlycooled to −10° C., there would be sufficient time for intracellularwater to diffuse out and enter the extracellular space along aconcentration gradient. This causes the intracellular soluteconcentration to rise and depress the intracellular melting/freezingtemperature, thereby helping to reduce the probability of lethalintracellular ice formation at colder temperatures. However, if the skinis triggered to freeze at −10° C. (with a large supercooling window),there will not be enough time for cell dehydration, and thus nointracellular freezing point depression. Therefore, at a coldersupercooling temperature (−10° C.), intracellular ice formation andassociated increased cell damage is more likely. A large amount ofsupercooling has been demonstrated to correlate with greater risk ofintracellular ice formation, which sometimes is desirable, but in otherinstances may be undesirable, depending on what tissue is and is notbeing targeted.

As discussed in connection with FIGS. 20A-20F and 21 , treatment methodsdisclosed herein can provide complete control over all freeze-thawparameters without allowing substantial skin supercooling that freezeslarger amounts of tissue in faster time periods than procedures that donot use supercooling. When the associated effects of increased tissuedisruption or damage in shorter time periods with supercooling is ofparticular therapeutic interest, a predetermined skin supercooling levelwith a predetermined duration can be achieved. A skin freeze can betriggered when a temperature of the applicator, coupling media, and skinis further cooled by, for example, a predetermined amount (e.g., lessthan 1° C., 2° C., 3° C., or 4° C.). For example, the applicator can beheld at a temperature slightly colder than the freezing point of theselected coupling media, which contains an ice-nucleating substance andfreezing point depressant, to ensure a layer of the coupling mediacontacting the applicator is frozen. By controlling the thickness of thelayer of coupling media, the temperature gradient across the couplingmedia layer can result in a temperature slightly warmer than itsfreezing point and thus unfrozen coupling media can remain in contactwith the skin. In some embodiments, the coupling media could be ahydrogel because hydrogels can be formulated to have precisethicknesses.

FIG. 22A shows a liquid coupling media that can serve as an insulatorfor ice inoculation of the skin, thereby allowing the skin to supercool.To trigger a skin freeze, the applicator can be cooled a few degreesfurther to freeze the entire volume of coupling media. As shown in FIG.22B, this process shifts the temperature profile to the left of thefreezing point of the coupling media. When ice crystals in the couplingmedia do reach the skin, the supercooled skin can freeze immediately orin a short period of time.

FIG. 23 shows freezing temperatures for varying concentrations of PG inwater. FIGS. 24A-24F show stages of a method for supercooling the skinto −13° C., holding the temperature for 3 minutes, and then initiating afreeze event at −15° C. using a coupling media comprising 26% by volumePG with a freezing temperature of about −11.5° C. The concentration ofthe PG solution can be selected based on the desired temperature forinitiating a freeze event.

Referring to FIGS. 24A-24F, coupling media can be applied to theapplicator and the skin. The applicator is cooled by ramping down atemperature of a temperature-controlled surface to freeze couplingmedia, and it is then heated by ramping up to −13° C. Holding thetemperature at −13° C. will ensure that at least a layer of frozen mediaremains in contact with the applicator. Coupling media in a liquid stateis applied to the skin surface. The applicator is applied to the liquidcoupling media on the skin and then cools the coupling media and theskin to freeze the target tissue. By selecting the composition of thecoupling media (e.g., concentrations of PG, glycerol, etc.),melting/freezing points can be selected which results in desiredtemperatures for supercooling the skin while providing both a thin layerof frozen coupling media (e.g., a frozen layer on the applicatorsurface) and a thin layer of liquid coupling media (e.g., a liquid layeron the skin surface).

FIG. 24A shows a layer of frozen coupling media 231 carried by theapplicator. A carrier in the form of a paper towel soaked with 26% byvolume PG/water can be placed on the temperature-controlled surface 211.The temperature-controlled surface 211 can be precooled to rapidlyfreeze the coupling media 231 once the paper towel is applied. In otherprocedures, coupling media is spread, sprayed, or otherwise applieddirectly to the temperature-controlled surface 211.

FIG. 24B shows another carrier in the form of a paper towel soaked with26% by volume PG/water (at room temperature) applied to the subject'sskin. The layer of liquid coupling media 233 (e.g., 26% PG/water) on theskin can be thick enough to prevent direct contact between the frozencoupling media 231 and the subject's skin upon placement of theapplicator. Additionally, the liquid coupling media 233 helps improve apatient's comfort when placing the frozen coupling media 231 upon thecoupling media 233.

FIG. 24C shows the applicator after the frozen coupling media 231 hasbeen placed in contact with the room temperature coupling media 233. Thethickness and temperature of the liquid coupling media 233 can beselected such that it will melt only part of the frozen coupling media231 so as to maintain a thin layer of frozen coupling media 231 alongthe temperature-controlled surface 211. A control system can control theapplicator to maintain an applicator surface temperature at a targettemperature, such as −13° C., which is below the freezing point (−11.5°C.) of 26% PG.

The applicator can continuously or intermittently extract heat togradually increase the volume of frozen coupling media for a holdingperiod. FIG. 24D shows the thickened layer of frozen coupling media 231.The coupling media 233 contacting the skin can remain in a liquid stateand remain near but below its freezing temperature of −11.5° C.

FIG. 24E shows the freezing front 221 moving toward the skin surface asthe coupling media is cooled. When the coupling media in contact withthe skin freezes, the supercooled skin will be inoculated and freezerapidly over a period of a few seconds. For example, the applicatortemperature can be ramped down to a temperature of about −15° C., −14°C., or −13° C. in order to freeze the entire coupling media volume(i.e., coupling media 231, 233), and thus trigger a freeze event in theskin.

The freeze event can be triggered by a temperature a few degrees colderthan the supercooling temperature. In one procedure, skin can be cooledto a supercooling temperature of −13° C. while still being able totrigger a freeze at a temperature only slightly lower, such as −15° C.This “dive” temperature of 2 degrees is much smaller than those requiredby conventional techniques that do not use a coupling media containingice crystals that contact the skin (which are of the order of about 10degrees) and serves as a skin-ice inoculating agent for triggering apredictable freeze event. For any given maximum end temperature for theapplicator, a smaller dive temperature can result in a largersupercooled volume, and at the time of the tissue freeze, the frozenvolume will also be larger compared to a treatment with a larger divetemperature. After the tissue has been frozen for a desired length oftime, the applicator can warm the tissue to inhibit or limit furtherdisruption, injury, etc. Warming and cooling cycles can be repeated anynumber of times in any order to thermally affect the targeted tissue.

FIG. 24F shows the entire volume of coupling media at theapplicator-skin interface and underlying tissue in a frozen state. Theapplicator can be cooled or heated to increase or decrease the volume offrozen tissue.

FIG. 25 is a plot of temperature versus time for supercooling andfreezing tissue. The skin can be cooled to −20° C., and then a freeze isinitiated at −25° C. by selecting a 37% by volume PG gel that has afreezing point of −19° C. Upon freezing of the skin, the applicator canrapidly warm the epidermis and hold it at a temperature sufficientlyhigh enough to keep the epidermis unfrozen. For example, the epidermiscan be held at a temperature of 1.5° C. or 2° C. This maximizes theunderlying dermal freeze exposure to increase dermal damage and limitsor minimizes the epidermal freeze exposure to reduce epidermal damage.Accordingly, warming can be used to minimize, limit, or substantiallyprevent thermal injuries that lead to hypopigmentation (skinlightening), hyperpigmentation (skin darkening), and/or otherundesirable effects.

The skin can be cooled to a temperature above the freezing point of thecoupling media in order to trigger a freeze event. When tissue issupercooled at −13° C. with a coupling media that has a slightly warmerfreezing temperature (e.g., a freezing temperature of −11.5° C.), theskin will not be inoculated at temperatures significantly warmer than−13° C. In some procedures, it may be desirable to initiate a skinfreeze at higher temperatures to minimize or limit injury to theepidermal tissue during the freeze event. To address this need, a highertemperature melting/freezing point can be achieved by dilution of thecoupling media to a lower concentration of a freezing point depressant.The melting/freezing temperature of the coupling media can be raised asufficient amount to trigger a freeze at a temperature well above thesupercooling temperature. Briefly, supercooling at time t1 can beaccomplished by choosing a coupling media that has a freezingtemperature lower than that at time t1. After supercooling, theapplicator temperature ramps up to a higher temperature at time t2. Avolume of water can be delivered into the coupling media to dilute it,ensuring that the diluted coupling media has a freezing point warmerthan the temperature at t2. This will trigger a freeze in the dilutedcoupling media to quickly trigger a skin freeze. A relatively warmon-command freeze in the supercooled diluted coupling media can betriggered using, for example, energy (e.g., ultrasound), low temperatureprobes (e.g., an extremely small cold finger probe), and/or an INA.

FIG. 26 is a plot of temperature versus time for a procedure that cyclestwice to supercool tissue and then triggers a freeze. Generally, tissuecan be cycled between two temperatures (e.g., −10° C. and −20° C.) tosupercool targeted tissue. A freeze event is triggered while at thehigher temperature (e.g., −10° C.) or another suitable temperature. Thefreezing point of the coupling media can be selected to ensure that thecoupling media does not freeze during a supercooling cycle. In someembodiments, the coupling media can comprise at least 39% by volume PGthat has a freezing point of −20.5° C. so that the coupling media doesnot freeze during a supercool cycle at −20° C. to avoid initiating apremature skin freeze.

At the end of the supercool cycle, the temperature of the applicator canbe raised to a higher temperature (e.g., −10° C.) suitable for iceinoculation. A substance, such as cold water at 1° C., can be infusedthrough the applicator to dilute the coupling media. The temperature andflow rate of the water can be selected such that the diluted couplingmedia has a freezing point warmer than a predetermined value. Forexample, the diluted coupling media can have freezing point higher thanabout −10° C. for freeze inoculation at about −10° C.

FIGS. 27A-27C show an applicator and a coupling media. Referring now toFIG. 27A, liquid coupling media is located along atemperature-controlled surface 243 of the applicator. Conduits, plates,and/or fluidic components of the applicator can have one or morethermally insulating coatings, layers, etc., to avoid unwanted freezeduring infusion because the cold applicator plate can be at relativelylow temperatures, for example, less than −5° C., −10° C., or −12° C.Additionally or alternatively, the applicator can include thermalelements (e.g., heating elements) for warming the diluting liquid,coolant (e.g., coolant delivered through the applicator), and otherworking fluids.

FIG. 27B shows the applicator and diluted coupling media. A dilutingliquid has passed through the conduit to dilute the coupling media. Theamount of diluting liquid can be selected to achieve the desiredcoupling media concentration. The coupling media can freeze as theapplicator is cooled and the temperature of the coupling mediastabilizes at a predetermined or target temperature, such as −8° C.,−10° C., or −12° C. An INA can be incorporated into the coupling mediaor into the infused liquid to promote freezing.

Ultrasound can be used to initiate, promote, and/or control a freezeevent. Referring now to FIG. 27A, cold water can be delivered into anultrasound chamber and, in some embodiments, can contact an uppersurface of the liquid coupling media. Cooling elements, cold plates, orother components of the applicator can cool the water surrounding anultrasound probe, which can be activated to deliver ultrasound energy tothe cooled water to cause freezing. Ultrasonic agitation (e.g.,ultrasonic agitation with an appropriate frequency, power, etc.) cangenerate an instantaneous freeze event such that the freeze propagatesthroughout the chamber and reaches the coupling media, therebytriggering a freeze in the diluted coupling media shown in FIG. 27B.

One or more INAs can be incorporated into the coupling media before,during, and/or after applying the coupling media to the patient.Dilution of the coupling media to a point where its diluted meltingtemperature is above its actual temperature will cause the dilutedcoupling media to freeze, which in turn will cause freezing of the skin.

FIGS. 28-31 show treatments that can involve supercooling. Supercoolcycling can cover a broad temperature range, with the coldest desiredsupercool temperature oftentimes being too cold to use as a freezingtemperature because it would cause excess damage to the epidermis.Coupling media that remains in a liquid state during the supercool cyclewill often not allow ice inoculation because the supercoolingtemperature range is above the freezing point of the coupling media.However, dilution of the coupling media raises the freezing point of thecoupling media and enables freeze inoculation of the skin at warmerapplicator and epidermal temperatures. Advantageously, epidermaltemperatures can be sufficiently warm to inhibit, limit, orsubstantially prevent hypopigmentation, hyperpigmentation, or otherundesirable effects.

Dilution also enables supercool cycling at relatively low temperatures(e.g., −10° C., −15° C., −20° C.) and tissue freezes at relatively hightemperatures (e.g., −10° C., −5° C., −4° C., −3° C., or −2° C.) toenhance or maximize damage to targeted tissue and limit or minimizedamage to non-targeted tissue. The targeted tissue can be tissue in thedermis and/or lower skin layers, and the non-targeted issue can be theepidermis or shallow tissue. Although enhancing or maximizing damage canbe achieved by multiple consecutive treatments with differentconcentrations of coupling media, a single treatment can provide desireddamage to reduce treatment time and costs.

FIG. 28 shows an applicator 261, media 262 with an INA, and a couplinglayer 263. An INA can be placed in direct contact with the skin surfaceto facilitate a predictable freeze of the skin. In some procedures, theINA is placed in direct contact with a cooling surface of theapplicator. For example, the media 262 can include one or more INAs(e.g., SNOMAX®/water mix). The coupling layer 263 can includecellulose-derived layer and solution, such as water, and can be indirect contact with a treatment site. In some embodiments, the INA canbe applied using a thin layer (e.g., paper) soaked with an INA couplingagent, mixed with a gel like substance, delivered via an deliveryinstrument (e.g., syringe), or sprayed over the surface. One skilled inthe art could substitute appropriate coupling layers materials,chemicals, conditions, and delivery systems with other materials,chemicals, conditions, delivery systems, etc.

FIG. 29 shows a plot of temperature versus time of a temperature profilefor triggering ice nucleation via INAs. Tissue can be supercooled to atemperature above the INA activation temperature. The temperature of theINA is then lowered to its activation temperature to initiate icenucleation to produce a partial or total freeze event (indicated by the“*”) propagating into and through the skin. The cycle can be completedby holding a temperature of the applicator to permit the growth of icecrystals at the treatment site. After completing the cycle, thetemperature of the applicator can be gradually raised at a desired thawrate to warm the skin.

FIG. 30 shows an applicator and an INA applied to a treatment site. TheINA can be delivered into coupling media 271, onto a subject's skinsurface, and/or into the skin to predictably trigger ice nucleation. Theapplicator can include embedded fluidics for controllably delivering theINA to the interface between the applicator and the subject's skin. Theinfused INA can be at a specific treatment temperature or within apredetermined temperature range to inhibit ice nucleation within, forexample, coupling media at the interface or the tissue itself. In otherembodiments, the INA is sprayed or otherwise delivered to the couplingmedia or the subject's skin. In some procedures, the applicator coolsthe coupling media and the subject's skin. The applicator can be liftedoff the coupling media, and the INA can be sprayed onto the cooledcoupling media. After spraying the INA, the applicator can be reappliedto the treatment site to continue cooling the INA, coupling media, andtissue. Needles, rollers, and other delivery instruments can be used toapply one or more INAs. Other techniques can be used to provide INAinfusion through the coupling media, as well as on or into the couplingmedia and/or subject's tissue, etc.

FIG. 31 shows a plot of temperature versus time for delivering an INAfor nucleation. The INA can be delivered to a treatment site, which canbe at or below the INA's activation temperature (indicated by a dashedline), at a specific time to initiate nucleation. As indicated by thearrow, the INA can be infused to initiate the freeze event. Accordingly,the INA's activation temperature can be selected to trigger a controlledfreeze event.

Various techniques can be used to protect non-targeted tissue whileaffecting targeted tissue volumes and/or specific structures within, forexample, the epidermis, dermis, subcutaneous tissue, etc. The targetedstructures can include, without limitation, hair (e.g., hair follicles),skin appendages (e.g., sweat glands, sebaceous glands, etc.), nerves,and/or dermal components, such as collagen, elastin, or bloodmicrovascularity. Targeted structures can be affected while inhibiting,preventing, or substantially eliminating unwanted side effects. Becauseappendages and other cells/structures may have different lethaltemperatures, a multi-step temperature profile can be used to targetspecific tissue and/or structures. Moreover, preserving the non-targetedtissue, such as the epidermis, from undue injury or damage could bebeneficial in to prevent, for example, pigment changes and/or scarring,as well as to promote healing. Freezing the epidermis at a differenttemperature than the underlying dermis can be achieved by using thecharacteristic activation temperature of the INA and by intentionallysupercooling the dermis at lower temperatures before applying the INA.In some procedures, the epidermis can be at a higher temperature toinhibit, limit, or substantially prevent permanent thermal injuries tothe epidermis.

Some embodiments of the technology include methods of using linkedpolymers containing water, a crosslinked polymer that contains water,optionally an INA, and/or optionally a freezing point depressant forcontrolled freeze of skin tissue. According to one preferred embodiment,the polymer can be a hydrogel for use for controlled freezing of skintissue. The hydrogel can be an effective initiator of a freeze event. Ashydrogel freezes, it can provide initial seeds or crystal sites toinoculate and freeze tissue, thus catalyzing a controlled predictablefreeze at specific temperature(s) in skin.

FIGS. 32A-32D show IR imaging of tissue freeze inoculation using ahydrogel with supercooled tissue. FIG. 32A shows skin tissue (dashedarea) over two hydrogels (left and right halves) and a cooling plateoverlying the hydrogels. FIG. 32B shows freezing at a lower leftportion. FIG. 32C shows freeze propagation along most of the lefthydrogel demonstrating ice inoculation. FIG. 32D shows freezepropagation through both hydrogels.

Referring now to FIG. 32A, the skin tissue at the area indicated bydashed lines contacts two half-sheets of hydrogel. A hydrogel sheet withno PG or other freezing point depressant is located to the left of thevertical line, and a hydrogel sheet with about 50% by volume PG islocated to the right of the vertical line. A rectangular cooling plateis located on the hydrogel sheets. The skin surface is in direct contactwith both hydrogels, which in turn are in direct contact with thecooling plate. In this manner, the hydrogels can be held firmly betweenthe patient and the applicator.

The images were generated after a few minutes of supercooling thetissue. The temperature of the supercooled tissue was lowered to atrigger temperature to trigger a freeze (illustrated in lighter color)in the non-PG hydrogel, illustrated on the left side of FIG. 32B. FIGS.32B and 32C show freeze propagation caused using only a hydrogel. Theright half of FIG. 32B shows a section of the 50% by volume PG hydrogeland adjacent skin that has not been frozen. FIG. 32C shows the freezepropagating across the hydrogel, through the tissue, and toward thehydrogel with the temperature depressant. This shows that varyingconcentrations of PG or other freezing point depressants can be includedin the hydrogel to lower the hydrogel's melting/freezing temperature toa desired value lower than 0° C. For water-based hydrogels with no PG orother freezing point depressant, freezing at temperatures close to 0° C.is inconsistent and unpredictable for a controlled heterogeneousnucleation. However, hydrogels used in combination with an INA providethe capability for a controlled freeze at subzero temperatures,including temperatures close to 0° C. (or lower when used in conjunctionwith a freezing point depressant) in a more predictable manner.

FIGS. 33A-33D are IR images showing tissue freeze inoculation usingcombined materials and the effect over time of placing an INA (e.g.,SNOMAX® or other suitable INA derived from bacterium PseudomonasSyringae) on a supercooled hydrogel to trigger a freeze. FIG. 33A showsplacement of a grain of an INA on a supercooled hydrogel. FIG. 33B showsan INA inoculating the hydrogel and a freeze starting to propagatearound the INA. FIG. 33C shows freeze propagating across the hydrogel tothe tissue demonstrating ice inoculation of skin. FIG. 33D shows thecompleted freeze propagation. Combined, the FIGS. 33A-33D show thefeasibility of using an INA as a seed for inoculating a controlledfreeze in skin tissue. Details of FIGS. 33A-33D are discussed below.

FIG. 33A shows unfrozen hydrogel and the INA being placed near an edgeof a cooling plate (indicated by dashed lines) while the cooling platecools the tissue. Upon initial placement of the INA, there is nosubstantial freezing through tissue facing the cooling plate. The INAcan initiate the freezing process by serving as an ice nucleator and canraise the predictable freezing temperature of water to about −3° C.Although the melting/freezing temperature of water is 0° C., water hasthe tendency to supercool, so its freezing temperature is often farlower than 0° C. or −3° C. when an INA has not been used. The INA caninitiate the freezing process by serving as an ice nucleator and canraise the predictable freezing temperature of water to about −3° C. TheINA can be selected to raise the predictable freezing temperature ofwater to other desired temperatures. When selecting an INA, one skilledin the art can choose appropriate agents (e.g., organic or inorganicderived agents) to use for specific desired temperature(s) and to bedelivered at specific times for a specific treatment purpose. Differenttechniques can be used to incorporate INA into hydrogels. For example,an INA can be sandwiched between layers of a hydrogel so as to betotally contained and encapsulated therein and so as to never come incontact with skin or tissue. An encapsulant can be disrupted, destroyed,or otherwise altered to release the INA. In some embodiments, a coolingplate of an applicator can deliver the INA to the hydrogel via one ormore needles, exit ports, or other delivery means. The INA can beapplied at a single location or multiple locations, or it can be mixedinto the hydrogel compound. Additionally, the INA can be inside amicrometric wall made of hard or soft soluble film so as to avoid directskin contact and have controlled degradation by passive or active means.Additionally, the INA can be injected or delivered into or onto theskin.

FIG. 33B shows the tissue after the freeze has propagated away from theINA. The frozen material is illustrated by a lighter color against adarker background color, which illustrates non-frozen tissue. FIG. 33Cshows freeze propagating across the hydrogel facing the cooling plate.FIG. 33D shows the completed freeze propagation to freeze all of theskin directly contacting the hydrogel.

FIGS. 34A and 34B are cross-sectional views of the cooling applicatorapplied to a treatment site. Referring now to FIG. 34A, only a hydrogelis located between the applicator and the subject's skin. The coolingapplicator can be disposed over a protective layer in the form of a thincover layer. The protective layer can be a liner or other component forpreventing cross-contamination or soiling by the hydrogel. As atemperature-controlled surface of the cooling applicator is cooled, itin turn cools the skin by withdrawing heat from the skin through thehydrogel and thin cover layer.

FIG. 34B shows a hydrogel and an INA located between the applicator andthe subject's skin. The INA can be a liquid, gel, cream, preformed sheetor layer located along a surface of the hydrogel. When the applicator isapplied to the hydrogel, the INA can be located at thehydrogel-applicator interface. The hydrogel and/or hydrogel/INA/freezingpoint depressant can be selected to melt/freeze at a specifictemperature designed into its formulation.

The hydrogel of FIGS. 34A and 34B can be formulated to have aconstituent ratio of water-monomer-crosslinker and/or other chemicals,such as one or more INAs, freezing point depressants, etc., to achieve aspecific freezing point (or close range of temperatures) that may or maynot allow for skin supercooling. An INA can have a known activationtemperature (natural freezing point) and can be in solid form (i.e.,powder) or mix solution with a desired concentration to create apredictable and consistent skin freeze. With such thermal couplingmaterials or compounds, a desired treatment temperature protocol oralgorithm can be implemented to allow for supercooling if desired, or nosupercooling if desired, and to allow for predictable and controlledfreezing at preferred temperatures and times.

FIG. 35 is a plot of temperature versus time for triggering a freezeagent in accordance with embodiments of the disclosed technology. Thetemperature profile, protocol, and/or algorithm for triggering one morefreezes allows supercooling of tissue at temperatures allowed by thehydrogel or hydrogel/INA. The temperature of the targeted tissue can bekept in the supercooling temperature range for a supercooling period.The temperature of the hydrogel is then lowered to a freezingtemperature to cause ice nucleation of the hydrogel or materials. Whenit undergoes a freeze event, the underlying targeted tissue can be at orslightly warmer than the hydrogel. In some procedures, both the hydrogeland targeted tissue are supercooled while the temperature of thetemperature-controlled surface of the applicator is held generallyconstant. In other procedures, the targeted tissue can be partiallyfrozen while the hydrogel is supercooled. Subsequent freezing of thehydrogel can cause further freezing of the target tissue until thedesired level of freezing in the targeted tissue is achieved. Othercoupling media can be used with the temperature profile shown in FIG. 35.

FIG. 36 shows an applicator positioned to produce a controlled freeze ina coupling media delivering the agent onto a surface of the couplingmedia, into the coupling media, or at another suitable location forinitiating a freeze event in the coupling media. The applicator caninclude one or more needles (e.g., a microneedle array), fluidcomponents (e.g., conduits, pumps, valves), reservoirs (e.g., reservoirsholding coupling media), or the like. In some embodiments, the agent isdelivered out an exit port at the bottom of a cooling plate of theapplicator.

FIG. 37 shows a cooling applicator with an external nucleating elementconfigured to initiate a freeze event external to an applicator-hydrogelinterface. The external nucleating element can include one or moreenergy emitting elements capable of initiating a freeze event. In someembodiments, an external nucleating element delivers energy (e.g.,ultrasound energy, RF energy, etc.) to an edge region of the couplingagent to produce a freeze, which propagates through the coupling agent,including a region of the coupling agent directly between the coolingapplicator and the tissue site. In other embodiments, a separatenucleating instrument can initiate nucleation and can be a probe with anucleating element.

FIG. 38 is a cross-sectional view of a cooling applicator that providesenergy-based activation. Coupling media, hydrogels, hydrogel/INAmixtures, or other materials for generating a controlled freeze can belocated between the cooling applicator and the treatment site. In someembodiments, an encapsulated ice nucleator can be part of or locatedwithin a layer of coupling media. Energy can break the encapsulation torelease the ice nucleator at a desired time. This can cause a freezeevent that spreads through the coupling media and into the surface ofthe skin. Once ice crystals contact the surface of the skin, the freezecan propagate through the skin. The actuation energy can be, withoutlimitation, mechanical energy (e.g., vibrations, ultrasound, etc.),electrical energy, and/or electromagnetic radiation (e.g., light).

FIGS. 39 shows a temperature profile for supercooling skin prior toinitiating a freeze event. Freeze activation can be controllablyinitiated to control freeze onset while a temperature-controlled surfaceof the applicator and/or the target tissue is held at a constant steadystate temperature. It may be desirable to supercool and freeze skin andsubcutaneous tissue for a specific time and at a specific temperature(or temperature range) to allow controlled supercooling of a tissuevolume that is sufficiently large to cause a widespread freeze upontissue inoculation.

It may be advantageous to cool tissue and/or affect specific structureswithin the dermis and subcutaneous tissue, like hair, skin appendages,nerves, dermal components such as collagen, elastin, or bloodmicrovascularity but at the same time to preserve the epidermis. Sinceappendages and other cells/structures may have a different lethal orinjury temperature, a multi-step temperature profile may be needed.Moreover, preserving the epidermis could be beneficial in the preventionof skin pigment changes and skin scarring. Additionally, preserving theepidermis can result in more favorable healing and fewer side effects.Freezing the epidermis at a different temperature than the underlyingdermis is possible by using the aforementioned techniques. Specifically,the skin bulk tissue can be supercooled at low temperatures and then thetemperature of the epidermis can be raised before, for example,delivering the INA or activating nucleation. Epidermal sensitivity isreduced when the epidermis freezes at a temperature of around −5° C. orhigher. If freezing below those temperatures occurs, melanocytes and/ortheir melanin production in the epidermis may get unduly altered causingpigmentation. So, according to some embodiments of the disclosedtechnology, temperature protocols can be used that cause freezing of theepidermis at or above −5° C.

FIG. 40 is a plot of temperature versus time where, after supercoolingand before freezing, a temperature of the applicator is adjusted to warmthe epidermis such that the applicator and/or epidermis is at a highertemperature (e.g., −6° C., −5° C., −4° C., etc.) than the supercoolingtemperature. After warming the epidermis, a freeze event is generated.For example, the temperature profile shows activation or delivery ofINAs at a warmer activation temperature suitable for protecting tissue,such as the epidermal or upper layers of the skin. The warming rate,activation temperature, and time period for the activation temperaturecan be selected based on the desired tissue protection and affects tothe targeted tissue. The activation holding time period can be increasedor decreased to increase or decrease protection of the epidermis.

FIG. 41 shows a plot of temperature versus time for a cooling protocoland three cross-sectional views of an applicator and skin tissuetemperature distributions. As shown in the plot, atemperature-controlled surface of an applicator can be held at −10° C.for 5 minutes (for supercooling) and then increased at a desired rate(e.g., 2° C./s, 2.5° C./s, etc.). A subsequent freeze event is shown bya rise of temperature after about time=400 seconds. Computationalmodeling (COMSOL) was used to generate the results. The model was athree-dimensional bioheat transfer model for the treatment of skin usinga cooling applicator and was used to generate plots discussed herein.

The images show temperature distributions in tissue related to thetemperature profile step change of the temperature-controlled surfacefrom −10° C. to −4° C. An isotherm has been added (T=0° C.) at time=380seconds, time=385 seconds, and time=400 seconds. The isotherm at T=0° C.is the boundary in which phase change to ice crystallization (freezingof skin) may extend the most (i.e., deeper tissue is warmer than 0° C.and will not freeze if ice nucleation were to occur since the fluid inthe skin at this depth is above its freezing temperature).

FIG. 42 shows a temperature profile in the epidermis/dermis (log scale)at time=380 seconds for an applicator at −10° C. showing the temperatureof T=0° C. at 2 mm depth into the skin. The depth of the T=0° C.isotherm is about 2 mm. Accordingly, the freeze may extend down to about2 mm into the skin at this time point if nucleation occurred at thistime point. The temperature gradient can be observed as well, showing agradient of T=−8° C. at the skin surface to 0° C. at a depth of 2 mm.

FIG. 43 shows a temperature profile in the epidermis/dermis (log scale)showing the temperature as applicator step-ups from −10° C. to −4° C. at2.5° C./s, with lines plotted for time=380 seconds, time=385 seconds,and time=400 seconds. The temperature profiles throughout the depth ofthe skin at time=380, time=385, and time=400 seconds, in other words,are both before and after the applicator temperature has transitionedfrom −10° C. to −4° C. Temperature gradients in the epidermis are above−5° C. at time=400 seconds so that a controlled freeze may be triggered.The epidermis will freeze at a more optimal temperature (>−5° C.) butwith a superior extent of the skin freeze down to a depth ofapproximately 2 mm.

FIG. 44 shows one method of creating a tissue freeze using a devicepositioned within a subject. The device can be a needle that injects asubstance at a specific location. An interior surface of the needle canbe coated to facilitate delivery of the substance. The exterior surfaceof the needle can be coated with an agent or substance for treatingtissue, targeted structures, etc. Other devices can be inserted into thesubject to produce freeze events.

The injected substance can include, without limitation, hydrogel,hydrogel/INA, partially frozen water, ice nucleators, combinationsthereof, or the like. An advantage of injecting an ice crystal orsubstance (e.g., an INA) that will create an ice crystal is that afreeze event will occur at a specific region. The freeze event can beinitiated in the dermis or other lower tissue layer and not in theepidermis. This limits or minimizes damage to the epidermis.Additionally, the epidermis can be warmed to a temperature close to orabove its melting/freeze temperature. In some embodiments, a freezeevent can be initiated in tissue below the dermis, such as insubcutaneous tissue. After producing the freeze event, the same ordifferent needle can inject additional substances into the tissue. Theadditional substances can include cryoprotective agents, liquids (e.g.,warm water or saline), or other substances that can effect therapy.

Multiple injections can be made to create multiple freeze events. Afirst substance can be delivered into tissue to create a first freezeevent, and a second substance can be delivered into other tissue toproduce a second freeze event. For example, the first substance can beadapted to completely freeze at a first target region, and the secondsubstance can be adapted to produce a partial freeze event at a secondtarget region spaced apart from the first target region. Differentlevels of freezing and severity of thermal injury can be achieved eventhough the first and second target regions are at the same temperature.In other treatments, the first and second target regions can be atdifferent temperatures, and the first and second substances can beselected based on those temperatures. In this manner, different types offreeze events can be generated at different locations.

With continued reference to FIG. 44 , substances with thermal couplingmaterials or nucleators having freezing points at higher temperaturesthan a freezing point of fluid in skin tissue may be usedsynergistically with the treatment cycle to produce an intentionalfreeze of the material and sequentially trigger freezing propagationinto the skin at yet higher temperatures. Non-targeted tissue can bewarmed by the applicator (e.g., via conduction), injected warm liquid,and/or energy (e.g., RF energy). Warming cycles can be performed to warmthe epidermis immediately after producing a freeze event that injurestarget structures, such as sebaceous gland. This can help preventvisible alterations (e.g., hyperpigmentation, hypopigmentation, etc.) tothe epidermis. The injectable substance can be delivered to and aroundthe sebaceous gland and freeze event can then be triggered by atemperature dive, dilution, energy, etc.

FIG. 45 is a flow diagram illustrating a method 350 in accordance withan aspect of the present technology. In block 352, coupling media can beapplied to the subject. In block 354, an applicator cools the tissue toa temperature suitable for a freeze event. For example, a skin surfacecan be lowered to a first temperature between about −2° C. and −40° C.to supercool shallow tissue. In some embodiments, the first temperaturecan be a temperature between −5° C. and −15° C., −5° C. and −20° C.,−10° C. and −30° C., or other suitable temperature range below afreezing temperature.

In block 356, the surface of the human subject's skin is heated anamount sufficient to raise the skin surface temperature from the firsttemperature to a second temperature, which can be a non-supercooledtemperature, while the targeted region remains in the supercooled state.For example, the epidermis can be heated to a temperature higher thanabout 0° C., higher than about 5° C., higher than about 10° C., higherthan about 20° C., higher than about 30° C., or higher than about 35° C.There can be a temperature gradient between the targeted tissue and theskin surface such that most of the non-targeted shallow tissue is at anon-supercooled temperature.

In block 356, the device of FIG. 44 can cause nucleation at the targetregion to cause at least some fluid and cells in the supercooled tissueto at least partially or totally freeze. Warmed cells residing at thesurface of the human subject's skin do not freeze. As such, cells at theskin surface can be protected without using a chemical cryoprotectant.However, chemical cryoprotectants can be used to inhibit or limithyperpigmentation or hypopigmentation. In some embodiments, a probe canbe inserted into the subject to cause nucleation of the supercooledtissue via a mechanical perturbation, ultrasound, or other suitablenucleation initiator. The freeze event can cause at least partialcrystallization of a plurality of gland cells in the target region. Theillustrated device of FIG. 44 is positioned to produce a freeze eventthat causes crystallization of cells in the sebaceous gland.

In block 358 of FIG. 45 , the supercooled tissue can be maintained inthe frozen state for a predetermined period of time longer than, forexample, about 10 seconds, 12 seconds, 15 seconds, 20 seconds, or othersuitable length of time sufficient to treat acne, improve a quality ofhair, treat hyperhidrosis, etc. In certain embodiments, the skin iscooled/heated to keep the targeted tissue in at least a partially ortotally frozen state for the predetermined time longer than about 10seconds, 12 seconds, 15 seconds, or 20 seconds.

Heat can be applied to warm epidermal cells to a temperature abovefreezing while glands in the dermis are at or near the supercooledtemperature. The step of applying heat can include warming a portion ofmost of the epidermal layer under the treatment device to a temperatureabove about 0° C., about 5° C., about 10° C., about 20° C., about 25°C., or about 32° C. Warming can be accomplished before, during, or afterthe freeze event. The subject's body heat, warm blood, or othermechanisms can naturally heat the epidermis to avoid or limit freezedamage to those cells.

If deeper tissue is not targeted, such tissue could be warmed usingfocused electrical currents, such as focused ultrasound or RF energy.Applicators can include one or more electrodes, transducers, or otherenergy-emitting elements. For example, an applicator can cool the skinsurface shown in FIG. 44 to supercool the tissue, including the dermis.The applicator can deliver RF energy or focused electrical currents tothe underlying non-targeted subcutaneous tissue to localize supercoolingto dermal tissue. A freeze event is then initiated in the supercooleddermal tissue.

The methods disclosed herein are capable of supercooling tissue withoutinitiating nucleation by cooling tissue at a relatively slow rate (e.g.,the temperature profile can cause a slow cooling of the tissue at thetarget region). For example, the rate of cooling can be either equal to,slower or faster than about 0.5° C., 1° C., 2° C., 3° C., 4° C., 5° C.,6° C., 7° C., 8° C., 9° C. or 10° C. per minute. A preferred rate ofcooling is about either 2° C., 4° C., or 6° C. per minute. Additionallyor alternatively, a treatment device can apply a generally constantpressure during cooling to the supercooled temperature range to avoidpressure changes that would cause inadvertent nucleation. In a furtherembodiment, the targeted tissue can be cooled while the patient is heldstill (e.g., without movement of the treatment site) to avoidmechanically disturbing the supercooled tissue and unintentionallycausing crystallization.

F. Suitable Computing Environments

FIG. 46 is a schematic block diagram illustrating subcomponents of acomputing device in the form of a controller suitable for the system 100of FIG. 3 in accordance with an embodiment of the disclosure. Thecomputing device 700 can include a processor 701, a memory 702 (e.g.,SRAM, DRAM, flash, or other memory devices), input/output devices 703,and/or subsystems and other components 704. The computing device 700 canperform any of a wide variety of computing processing, storage, sensing,imaging, and/or other functions. Components of the computing device 700may be housed in a single unit or distributed over multiple,interconnected units (e.g., through a communications network). Thecomponents of the computing device 700 can accordingly include localand/or remote memory storage devices and any of a wide variety ofcomputer-readable media.

As illustrated in FIG. 46 , the processor 701 can include a plurality offunctional modules 706, such as software modules, for execution by theprocessor 701. The various implementations of source code (i.e., in aconventional programming language) can be stored on a computer-readablestorage medium or can be embodied on a transmission medium in a carrierwave. The modules 706 of the processor can include an input module 708,a database module 710, a process module 712, an output module 714, and,optionally, a display module 716.

In operation, the input module 708 accepts an operator input 719 via theone or more input/output devices described above with respect to FIG. 3, and communicates the accepted information or selections to othercomponents for further processing. The database module 710 organizesrecords, including patient records, treatment data sets, treatmentprofiles and operating records and other operator activities, andfacilitates storing and retrieving of these records to and from a datastorage device (e.g., internal memory 702, an external database, etc.).Any type of database organization can be utilized, including a flat filesystem, hierarchical database, relational database, distributeddatabase, etc.

In the illustrated example, the process module 712 can generate controlvariables based on sensor readings 718 from sensors (e.g., sensor 167 ofFIG. 2 ) and/or other data sources, and the output module 714 cancommunicate operator input to external computing devices and controlvariables to the controller 114 (FIG. 3 ). The output signals 720 can beused to control one or more applicators applied to the patient. In someembodiments, the output signals 720 can be commands for controllingapplicators. The display module 816 can be configured to convert andtransmit processing parameters, sensor readings 818, output signals 720,input data, treatment profiles, and prescribed operational parametersthrough one or more connected display devices, such as a display screen,printer, speaker system, etc. A suitable display module 716 may includea video driver that enables the controller 114 to display the sensorreadings 718 or other status of treatment progression.

In various embodiments, the processor 701 can be a standard centralprocessing unit or a secure processor. Secure processors can bespecial-purpose processors (e.g., reduced instruction set processor)that can withstand sophisticated attacks that attempt to extract data orprogramming logic. The secure processors may not have debugging pinsthat enable an external debugger to monitor the secure processor'sexecution or registers. In other embodiments, the system may employ asecure field programmable gate array, a smartcard, or other securedevices.

The memory 702 can be standard memory, secure memory, or a combinationof both memory types. By employing a secure processor and/or securememory, the system can ensure that data and instructions are both highlysecure and sensitive operations such as decryption are shielded fromobservation. The memory 702 can contain executable instructions forcooling the surface of the subject's skin to a temperature andcontrolling treatment devices in response to, for example, detection ofsupercooling, a partial or complete freeze event, movement of theapplicator (e.g., applicator pull off), or the like. In someembodiments, the memory 702 can include nucleation instructions that,when executed, cause the controller to command an applicator to alterthe composition of a coupling media, inject nucleation initiator, etc.Additionally or alternatively, the memory 702 can include thawinginstructions that, when executed, causes the controller to control theapplicator to heat tissue. In some embodiments, the stored instructionscan be executed to control the applicators to perform the methodsdisclosed herein without causing undesired effects, such assignificantly lightening or darkening skin one of more days after thefreeze event ends. The instructions can be modified based on patientinformation and treatments to be performed. Other instructions andalgorithms (including feedback control algorithms) can be stored andexecuted to perform the methods disclosed herein.

In some embodiments, the controller 114 is programmed to cause theapplicator to create or maintain at least one ice crystal and to inducea freeze event. The memory 702, for example, can contain instructionsthat when executed cause the applicator to operate to cause one or moreice crystals to contact the subject skin so as to induce a freeze event.In one embodiment, the memory 702 contains instructions that whenexecuted by the processor 701 cause the applicator to be a suitabletemperature for supercooling target tissue and for freezing the skinwithout lowering a temperature of the temperature-controlled surfacebelow a particular level. The instructions can be used to control orcommunicate with components of applicators. These components caninclude, without limitation, one or more thermoelectric elements, fluidelements, energy-emitting elements, and sensors. The thermoelectricelements can be Peltier devices capable of selectively cooling orheating the tissue. The fluid elements can be cooling channels,conduits, or other fluid elements through which fluid can flow to heatand/or cool tissue. The energy emitting elements can be radiofrequencyelectrodes, ultrasound electrodes, or other elements capable ofdelivering energy to control freezing, warm tissue, or the like.

Suitable computing environments and other computing devices and userinterfaces are described in commonly assigned U.S. Pat. No. 8,275,442,titled “TREATMENT PLANNING SYSTEMS AND METHODS FOR BODY CONTOURINGAPPLICATIONS,” which is incorporated herein in its entirety byreference.

G. Conclusion

The treatment systems, applicators, and methods of treatment can be usedto treat acne, hyperhidrosis, wrinkles, subcutaneous tissue, structures(e.g., structures in the epidermis, dermis, subcutaneous fat, muscle,nerve tissue, etc.), and so on. Methods for cooling tissue and relateddevices and systems in accordance with embodiments of the presentinvention can at least partially address one or more problems associatedwith conventional technologies as discussed above and/or other problemswhether or not such problems are stated herein. Methods for affectingskin of a human subject's body include positioning an applicator of acooling apparatus on the subject and removing heat from a treatment siteto affect the appearance of the subject's skin with or without causingan appreciable reduction of subcutaneous adipose tissue. Acne along theface can be treated without causing any reduction of subcutaneousadipose tissue wherein acne along the back can be treated while reducingof subcutaneous adipose tissue. Systems, components, and techniques forreducing subcutaneous adipose tissue are disclosed in U.S. Pat. No.7,367,341 titled “METHODS AND DEVICES FOR SELECTIVE DISRUPTION OF FATTYTISSUE BY CONTROLLED COOLING” to Anderson et al., U.S. PatentPublication No. U.S. 2005/0251120 titled “METHODS AND DEVICES FORDETECTION AND CONTROL OF SELECTIVE DISRUPTION OF FATTY TISSUE BYCONTROLLED COOLING” to Anderson et al., and U.S. Patent Publication No.2007/0255362 titled “CRYOPROTECTANT FOR USE WITH A TREATMENT DEVICE FORIMPROVED COOLING OF SUBCUTANEOUS LIPID-RICH CELLS,” the disclosures ofwhich are incorporated herein by reference in their entireties. Forexample, sufficient amount of thermal energy can be removed from thesite so as to reduce wrinkles by, for example, reducing the number ofvisible wrinkles and/or sizes of the wrinkles. In other embodiments, asufficient amount of thermal energy is removed from the treatment siteso as to tighten skin at the treatment site, or in further embodiments,to alter the tissue between a surface of the skin and subcutaneouslipid-rich cells of the subject's body. In a further embodiment, tissueis cooled to induce fibrosis that increases the firmness of tissue atthe treatment site. Fibrosis can be induced in the epidermis, dermis,and/or subcutaneous tissue. Vacuum applicators can stretch, stress,and/or mechanically alter skin to increase damage and fibrosis in theskin, affect glands, control freeze events (including initiating freezeevents), etc.

It will be appreciated that some well-known structures or functions maynot be shown or described in detail so as to avoid unnecessarilyobscuring the relevant description of the various embodiments. Althoughsome embodiments may be within the scope of the technology, they may notbe described in detail with respect to the figures. Furthermore,features, structures, or characteristics of various embodiments may becombined in any suitable manner. The technology disclosed herein can beused for improving skin and skin conditions and to perform theprocedures disclosed in U.S. Provisional Application Ser. No.61/943,250, filed Feb. 21, 2014, U.S. Pat. No. 7,367,341 entitled“METHODS AND DEVICES FOR SELECTIVE DISRUPTION OF FATTY TISSUE BYCONTROLLED COOLING” to Anderson et al., and U.S. Patent Publication No.U.S. 2005/0251120 entitled “METHODS AND DEVICES FOR DETECTION ANDCONTROL OF SELECTIVE DISRUPTION OF FATTY TISSUE BY CONTROLLED COOLING”to Anderson et al., the disclosures of which are incorporated herein byreference in their entireties. The technology disclosed herein cantarget tissue for tightening the skin, improving skin tone or texture,eliminating or reducing wrinkles, or increasing skin smoothness asdisclosed in U.S. Provisional Application Ser. No. 61/943,250.

Unless the context clearly requires otherwise, throughout thedescription, the words “comprise,” “comprising,” and the like are to beconstrued in an inclusive sense as opposed to an exclusive or exhaustivesense; that is to say, in a sense of “including, but not limited to.”Words using the singular or plural number also include the plural orsingular number, respectively. Use of the word “or” in reference to alist of two or more items covers all of the following interpretations ofthe word: any of the items in the list, all of the items in the list,and any combination of the items in the list. In those instances where aconvention analogous to “at least one of A, B, and C, etc.” is used, ingeneral such a construction is intended in the sense of the convention(e.g., “a system having at least one of A, B, and C” would include butnot be limited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense of the convention (e.g., “ a system having atleast one of A, B, or C” would include but not be limited to systemsthat have A alone, B alone, C alone, A and B together, A and C together,B and C together, and/or A, B, and C together, etc.).

Any patents, applications and other references, including any that maybe listed in accompanying filing papers, are incorporated herein byreference. Aspects of the described technology can be modified, ifnecessary, to employ the systems, functions, and concepts of the variousreferences described above to provide yet further embodiments. While theabove description details certain embodiments and describes the bestmode contemplated, no matter how detailed, various changes can be made.Implementation details may vary considerably, while still beingencompassed by the technology disclosed herein. The various aspects andembodiments disclosed herein are for purposes of illustration and arenot intended to be limiting, with the true scope and spirit beingindicated by the following claims.

What is claimed is:
 1. A method for treating a subject's skin,comprising: applying a coupling media to the skin, wherein the couplingmedia includes a freezing point depressant and either a liposome, anoil-in-water emulsion, a water-in-oil emulsion, or an oil-in-oilemulsion containing the freezing point depressant to enhance delivery ofthe freezing point depressant to the skin; and cooling the couplingmedia and a surface of the skin with an applicator to a temperaturebelow 0° C. to treat the skin.
 2. The method of claim 1, wherein thefreezing point depressant comprises propylene glycol, glycerol, and/orpolyethylene glycol.
 3. The method of claim 1, wherein the couplingmedia includes the liposome, and wherein the liposome and freezing pointdepressant is a propylene glycol liposome.
 4. The method of claim 1,wherein the coupling media includes the liposome, and wherein theliposome contains water such that the freezing point depressant and/orthe water are released into the skin after the liposome breaks down. 5.The method of claim 1, wherein the coupling media includes the liposome,wherein the method further comprises breaking a lipid encapsulation ofthe liposome using ultrasound energy, temperature cycling, and/or acleansing agent to release the freezing point depressant.
 6. The methodof claim 1, further comprising controlling cooling of the coupling mediaand the skin surface so as to not to freeze the epidermis.
 7. The methodof claim 1, further comprising controlling cooling of the coupling mediaand the surface cooling so as to freeze the skin.
 8. The method of claim1, wherein the coupling media further contains an ice nucleation agent,and wherein the method further comprises controlling cooling of thecoupling media and the surface of the skin to predictably freeze theskin based on nucleation caused by the ice nucleation agent.
 9. Themethod of claim 1, wherein the coupling media is either the oil-in-wateremulsion or the water-in-oil emulsion.
 10. The method of claim 9,wherein the oil-in-water emulsion or the water-in-oil emulsion is ananoemulsion.
 11. A method for treating a subject's skin, comprising:applying a coupling media to a surface of the subject's skin such thatan ice nucleating agent contained in the applied coupling media does notdirectly contact epidermal cells for a period of time; applying anapplicator to the subject's skin; and cooling the coupling media and thesurface of the skin with the applicator to lower a temperature of thesurface of the skin below 0° C. to cause a freeze event initiated by theice nucleating agent so as to freeze the skin.
 12. The method of claim11, wherein cooling the coupling media and the surface of the skininclude removing heat from the skin to start freezing the skin after theperiod of time.
 13. The method of claim 11, wherein the coupling mediacomprises a liposome, an oil-in-water emulsion, a water-in-oil emulsion,an oil-in-oil emulsion, and/or nano-emulsion.
 14. The method of claim11, wherein the coupling media includes a freezing point depressant forlowering a freezing point of the skin.
 15. The method of claim 14,wherein the freezing point depressant is configured to penetrate theskin only a shallow depth to lower a freezing point of epidermal tissuemore than dermal tissue.
 16. The method of claim 11, further comprisingbreaking apart a liposome of the coupling media to release the icenucleating agent for initiating the freeze event.
 17. The method ofclaim 11, wherein cooling the coupling media and the surface of the skinincludes: removing heat from the skin to supercool targeted tissue inthe skin; freezing the targeted tissue; and after freezing the targetedtissue, removing heat from and/or applying heat to the skin to keep anepidermal layer frozen while a dermal layer thaws and refreezes.
 18. Themethod of claim 11, further comprising either: diluting the couplingmedia to raise its freezing point to produce the freeze event, and/ordelivering energy to activate the ice nucleating agent, and/or reducingthe temperature of the coupling media to cause the freeze event after afluid in the subject's skin has been cooled below its freezing point.19. A system for treating a subject, comprising: an applicatorconfigured to reduce a temperature of a target region beneath a surfaceof the subject's skin to reduce the temperature of target tissue from anatural body temperature to a temperature for freezing the targettissue; a coupling media including a freezing point depressant andeither a liposome, an oil-in-water emulsion, a water-in-oil emulsion, oroil-in-oil emulsion containing the freezing point depressant to enhancedelivery of the freezing point depressant to the skin; and a controllerprogrammed to cause the applicator to cool the coupling media and asurface of the skin to a temperature below 0° C. to freeze the skin fora period of time.